β-substituted β-amino acids and analogs as chemotherapeutic agents

ABSTRACT

β-Substituted β-amino acids, β-substituted β-amino acid derivatives, and β-substituted β-amino acid analogs and (bio)isosteres and their use as chemotherapeutic agents are disclosed. The β-substituted β-amino acid derivatives and β-substituted β-amino acid analogs and (bio)isosteres are selective LAT1/4F2hc substrates and exhibit rapid uptake and retention in tumors expressing the LAT1/4F2hc transporter. Methods of synthesizing the β-substituted β-amino acid derivatives and β-substituted β-amino acid analogs and methods of using the compounds for treating cancer are also disclosed. The β-substituted β-amino acid derivatives and β-substituted β-amino acid analogs exhibit selective uptake in tumor cells expressing the LAT1/4F2hc transporter and accumulate in cancerous cells when administered to a subject in vivo. The β-substituted β-amino acid derivatives and β-substituted β-amino acid analogs and (bio)isosteres exhibit cytotoxicity toward several tumor types.

This application is a continuation of U.S. application Ser. No.15/182,851, filed on Jun. 15, 2016, now allowed, which is a continuationof U.S. application Ser. No. 14/613,143, filed on Feb. 3, 2015, nowissued as U.S. Pat. No. 9,394,237, which claims benefit under 35 U.S.C.§ 119(e) of U.S. Provisional Application No. 61/935,246, filed on Feb.3, 2014, each of which is incorporated by reference in its entirety.

FIELD

Disclosed herein are β-substituted β-amino acids, β-substituted β-aminoacid derivatives, and β-substituted β-amino acid analogs and their useas therapeutic agents. The β-substituted β-amino acid derivatives andβ-substituted β-amino acid analogs are selective substrates forLAT1/4F2hc and exhibit rapid uptake and retention in tissue such astumors expressing the LAT1/4F2hc transporter. Pharmaceuticalcompositions comprising the β-substituted β-amino acid derivatives andβ-substituted β-amino acid analogs and uses thereof are also disclosed.

BACKGROUND

The ability to selectively target chemotherapy has immense value inclinical practice. Cancer is a leading cause of death in the developedworld, with one in every three people developing cancer during his orher lifetime. There are many treatment options for cancer includingsurgery, chemotherapy, radiation therapy, immunotherapy, and monoclonalantibody treatment. Unfortunately, for many patients cancer treatmentoptions are limited and response rates remain low.

Surgery is the oldest effective form of tumor therapy and can oftenresult in a complete cure, depending of the type and nature of thetumor. Many tumors, however, occur in locations and/or number that makesurgery impossible or impractical. Also, surgical debulking is notguaranteed to remove all abnormal cells, particularly in the case oftumors located in the brain where maximum preservation of normal tissueis desired. Residual abnormal cells pose an increased risk of tumorre-growth and/or metastasis.

Radiation therapy is often used as an adjunct to surgery. Various typesof radiation, both from external and implanted sources, have been usedwith some success. Low linear-energy-transfer (LET) sources, such asβ-particles and γ-rays, require repeated treatments over extendedperiods of time to produce any significant reduction in tumor cells.High LET sources, such as neutrons, protons or α-particles, do notrequire oxygen to enhance their biological effectiveness. External beamtherapy has been available for decades, however, significant radiationdamage occurs to normal tissues, and patients often succumb towidespread radiation-induced necrosis (Laramore, et al., Cancer, 1978,42(1), 96-103).

Chemotherapy is used in attempts to cure or palliate cancer. Smallmolecule chemotherapeutics target rapidly dividing cells, halting cellproliferation by interfering with DNA replication, cytoskeletalrearrangements and/or signaling pathways that promote cell growth.Disruption of cell division slows the growth of malignant cells and mayalso kill tumor cells by triggering apoptosis. Alkylating agents, suchas bis(2-chloroethyl)amine derivatives, act by covalent interaction withnucleophilic heteroatoms in DNA or proteins. It is believed that thesedifunctional agents are able to crosslink a DNA chain within a doublehelix in an intrastrand or interstrand fashion, or to crosslink betweenDNA, proteins or other vital macromolecules. The crosslinking results ininhibitory effects on DNA replication and transcription with subsequentcell death. Since these drugs also indiscriminately kill normalpopulations of rapidly proliferating cells, such as those found in theimmune system and in the gastrointestinal tract, side effects that limittolerated doses, are common.

The harsh side effects and the ultimate failure of most chemotherapyregimens have motivated investigation of alternatives, including drugsthat target specifically tumor cells. Normal cells and tumor cellsdiffer markedly in nutrient and energy metabolism, a phenomenon known asthe Warburg effect (Ganapathy, et al., Pharmacol Ther, 2009, 121(1),29-40; and Vander Heiden, et al., Science, 2009, 324(5930), 1029-1033).Enhanced proliferation in tumor cells places increased demand fornutrients to serve as building blocks for the biosynthesis ofmacromolecules and as sources of energy. Tumor-selective nutrientaccumulation is most clearly evident in imaging studies of human tumorsusing positron emission tomography (PET) and [¹⁸F]-fluorodeoxyglucose(FDG). FDG accumulates at high levels in many kinds of solid tumors andis thought to be taken up into tumor cells by sugar transporters. Aminoacids are the primary source of cellular nitrogen, used for nucleotide,glutathione, amino sugar, and protein synthesis. In addition, tumorsoften utilize the carbon skeletons of amino acids as an oxidative fuelsource for ATP generation in addition to glucose and fatty acids(Baggetto, Biochimie, 1992, 74(11), 959-974; Mazurek and Eigenbrodt,2003, Anticancer Res, 2003, 23(2A), 1149-1154; and DeBerardinis, et al.,Proc Natl Acad Sci USA, 2007, 104(49), 19345-19350). Therefore, tumorcells must express select specific transporters to satisfy maintenanceand growth requirements for nutritional amino acids. To compete withsurrounding tissue for nutrients, tumor cells up-regulate levels ofcertain transporters to allow for more efficient extraction of nutrientsthan that of the host tissue.

Amino acid transport across the plasma membrane in mammalian cells ismediated by different transport “systems” such as the sodium-dependentsystems A, ASC and N, and sodium-independent system L (Christensen, PhysRev, 1990, 70, 43-77). System L is a ubiquitous plasma membrane aminoacid transport system that is characterized by the sodium-independentuptake of bulky, hydrophobic amino acids and its high affinityinteraction with 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH).System L activity is presently attributed to four sodium-independenttransporters (LAT1-4). However, most cancers over-express only onemember, the large amino acid transporter 1 (LAT1/4F2hc). Thistransporter is a heterodimer consisting of a light chain (LAT1) thatconstitutes the transporter and a heavy chain 4F2hc (also known as CD98,or Tumor Antigene TA1) that is required for proper targeting of thelight chain to the plasma membrane. The expression and activity ofLAT1/4F2hc correlates with cell proliferation and cancer growth; andup-regulation of LAT1/4F2hc has been observed, for example, in cancersof brain, colon, lung, liver, pancreas, and skin (Jager, et al., J NuclMed, 1998, 39(10), 1736-1743; Ohkame, et al., J Surg Oncol, 2001, 78(4),265-267; Tamai, et al., Cancer Detect Prev, 2001, 25(5), 439-445; Kim,et al., Anticancer Res, 2004, 24(3a), 1671-1675; Kobayashi, et al.,Neurosurgery, 2008, 62(2), 493-503; Imai, et al., Histopathology, 2009,54(7), 804-813; and Kaira, et al., 2009, Lung Cancer, 66(1), 120-126).Furthermore, the expression of LAT1/4F2hc has been used as anindependent factor to predict poor prognoses in patients with astrocyticbrain tumors, lung cancer, and prostate cancer (Nawashiro, et al., Int JCanc, 2006, 119(3), 484-492; Kaira, et al., Lung Cancer, 2009, 66(1),120-126; Kaira, et al., Cancer Sci, 2008, 99(12), 2380-2386; and Sakata,et al., Pathol Int, 2009, 59(1), 7-18). Inhibition ofLAT1/4F2hc-mediated transport with non-metabolizable amino acids such asBCH can reduce growth and induce apoptosis in cancer cells in vitro(Kim, et al., Biol Pharm Bull, 2008, 31(6), 1096-1100; Shennan andThomson, Oncol Rep, 2008, 20(4), 885-889; and Kaji, et al., Int JGynecol Cancer, 2010, 20(3), 329-336). Clinical studies have shown thatthe specificity and positive predictive value ofL-[3-¹⁸F]-α-methyltyrosine ([¹⁸F]-FAMT) PET is superior to [¹⁸F]-FDGPET. The uptake of [¹⁸F]-FAMT in tumors has been closely correlated withLAT1 expression (Haase, et al., J Nucl Med, 2007, 48(12), 2063-2071;Kaira, et al., Clin Cancer Res, 2007, 13(21), 6369-6378; and Urakami, etal., Nucl Med Biol, 2009, 36(3), 295-303).

In particular, melphalan is an effective chemotherapy drug used intreating multiple myeloma, ovarian cancer, retinoblastoma, and otherhematopoietic tumors. However, substrates such as gabapentin arereported to be transported much more rapidly than melphalan (Uchino, etal., Mol Pharmacol 2002, 61(4), 729-737). It is widely believed thatuptake of melphalan (Alkeran®, otherwise known as L-PhenylalanineMustard, or L-PAM) into cells is mediated by amino acid transporters.Melphalan is an alkylating agent linked to the essential amino acidphenylalanine. Because normal cells and tumor cells differ markedly innutrient and energy metabolism (Warburg effect) (Vander Heiden, et al.,Science, 2009, 324(5930), 1029-1033), melphalan was introduced intoclinical practice with the expectation that it would preferentiallyaccumulate in rapidly dividing tumor cells compared to normal cells,thereby increasing its overall therapeutic index. Surprisingly,melphalan caused many of the same side effects as other conventionalalkylation agents, including myelosuppression. In a series ofpublications, Vistica et al. examined melphalan transport in differentcell types and identified two independent transport systems formelphalan. One system, presumed to be System L, is characterized by thesodium-independent uptake of bulky, hydrophobic amino acids and itssensitivity toward inhibition with2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) (Vistica, BiochimBiophys Acta, 1979, 550(2), 309-317). A second transport system issodium-dependent, exhibits its highest affinity for leucine, but isinsensitive to both BCH and the system A-specific inhibitorα-amino-isobutyric acid (AlB) (Vistica, Biochim Biophys Acta, 1979,550(2), 309-317). Although LAT1 is overexpressed on the cell surface ofalmost all tumor cells regardless of the tissue of origin, responserates to melphalan are low for most cancer types, and the drug is onlyapproved for the treatment of multiple myeloma and ovarian cancer.Melphalan is a poor substrate for LAT1 compared to other large aminoacids such as phenylalanine or leucine (Uchino, et al., Mol Pharmacol2002, 61(4), 729-737; and Hosoya, et al., Biol Pharm Bull, 2008, 31(11),2126-2130). Nitrogen mustard derivatives with higher selectivity towardthe LAT1/4F2hc system could reduce side effects associated with nitrogenmustard therapy, allow for an increase in dose, and extend the use intoother areas of cancer treatment.

Although the potential for active transport strategies for increasingdrug uptake into tumor cells is known and generally accepted,chemotherapeutics and tumor imaging agents have in general not beenoptimized for transporters known to be over-expressed in tumor cells.While the general concept of using LAT1/2Fhc-selective compounds todeliver therapeutic agents to tumors is appreciated, the existing artgives no guidance as to how one prepares a composition that exploitsLAT1/4F2hc selective compounds. Thus, there is a need for newtherapeutic agents that are more selective toward LAT1/4F2hc.

Several amino acid-related drugs that are substrates of the LAT1/4F2hctransporter are known including L-Dopa, 3-O-methyldopa, droxidopa,carbidopa, 3,3′,5′-triiodothyronine, thyroxine, gabapentin, andmelphalan (Uchino, et al., Mol Pharm 2002, 61(4), 729-737; and del Amoet al., Eur J Pharm Sci, 2008, 35(3), 161-174).

SUMMARY

Differentiation of malignant cancer tissue from neighboring nonmalignanttissue can be accomplished by exploiting changes in biochemical fluxesthat occur in response to metabolic, genetic, and/or microstructuralchanges in the malignant cells. Compounds provided by the presentdisclosure substantially improve chemotherapy of tissue expressing theLAT1/4F2hc transporter including malignant tumors. The β-substitutedβ-amino acid derivatives and β-substituted β-amino acid analogs providedby the present disclosure provide greater uptake selectivity for thetarget tissue or cells expressing the LAT1/4F2hc transporter with lownon-specific uptake for non-target tissues or cells.

Embodiments provided by the present disclosure provide novelβ-substituted β-amino acid derivatives and β-substituted β-amino acidanalogs, and methods of using such derivatives, for example, aschemotherapeutic agents. Certain embodiments further relate to methodsof synthesizing β-substituted β-amino acid derivatives and β-substitutedβ-amino acid analogs and to pharmaceutical compositions comprising suchderivatives. The β-substituted β-amino acid derivatives andβ-substituted β-amino acid analogs the present disclosure exhibitselectivity for LAT1/4F2hc and therefore accumulate in cancerous cellswhen administered to a subject in vivo. Advantages provided by compoundsof the present disclosure reflect the properties of LAT1/4F2hcsubstrates, namely, blood brain-barrier (BBB) permeability, rapiduptake, and prolonged retention in tumors expressing the LAT1/4F2hctransporter, and further serve as chemotherapeutic agents.

Compounds

In a first aspect, compounds of Formula (1) are provided:

or a pharmaceutically acceptable salt thereof, wherein:

at least one of R¹ and R⁵ is independently selected from halogen,—N(R¹⁰)₂, —N⁺(—O⁻) (R¹⁰)₂, —N(OR¹⁰)(R¹⁰), —NO₂, —NO, —N(R¹⁰)(S(═O)R¹⁰),—N(R¹⁰)(S(═O)₂R¹⁰), —N(R¹⁰)(C(O)R¹⁰), —N(R¹⁰)(C(O)OR¹⁰),—N(R¹⁰)(C(O)N(R¹⁰)₂, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, —SH, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, —S(O)N(R¹⁰)₂,—S(O)₂N(R¹⁰)₂, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₃₋₆cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy, substitutedC₃₋₆ cycloalkyloxy, C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂cycloalkylalkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl,substituted C₇₋₁₆ arylalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ heteroalkoxy, substituted C₁₋₆ heteroalkoxy, C₃₋₆heterocycloalkyl, substituted C₃₋₆ heterocycloalkyl, C₄₋₁₂heterocycloalkylalkyl, substituted C₄₋₁₂ heterocycloalkylalkyl, C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₆₋₁₆ heteroarylalkyl, andsubstituted C₆₋₁₆ heteroarylalkyl;

one of R¹, R², R³, R⁴, and R⁵ comprises a chemotherapeutic moiety;

each of the other of R¹, R², R³, R⁴, and R⁵ is independently selectedfrom hydrogen, deuterio, halogen, —OH, —N(R¹⁰)₂, —NO₂, —NO, —CN,—COOR¹⁰, —CON(R¹⁰)₂, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₆ cycloalkyl,substituted C₃₋₆ cycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy,substituted C₁₋₆ heteroalkoxy, C₄₋₈ cycloalkylalkyl, and C₄₋₈cycloalkylheteroalkyl;

R⁶ is selected from a carboxylic acid (—COOH), a carboxylic acid analog,and a carboxylic acid (bio)isostere;

each R⁷ is independently selected from hydrogen, deuterio, halogen,hydroxyl, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, and phenyl; or two R⁷together with the carbon to which they are bonded form a ring selectedfrom a C₃₋₆ cycloalkyl ring and a C₃₋₆ heterocycloalkyl ring;

R⁸ is selected from hydrogen, deuterio, halogen, C₁₋₆ alkyl, substitutedC₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy,substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy, substituted C₁₋₆heteroalkoxy, C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, substituted C₃₋₆ cycloalkyloxy, —OH, —COOR¹⁰, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₆ cycloalkyl, and phenyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyland C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

L is —(X)_(a)—, wherein,

-   -   each X is independently selected from a bond (“—”), —C(R¹⁶)₂—,        wherein each R¹⁶ is independently selected from hydrogen,        deuterio, halogen, hydroxyl, C₁₋₄ alkyl and C₁₋₄ alkoxy, or two        R¹⁶ together with the carbon to which they are bonded form a        C₃₋₆ cycloalkyl ring or a C₃₋₆ heterocycloalkyl ring, —O—, —S—,        —SO—, —SO₂—, —CO—, and —N(R¹⁷)—, wherein R¹⁷ is selected from        hydrogen and C₁₋₄ alkyl; and    -   a is selected from 0, 1, 2, 3, and 4.

In a second aspect, chemotherapeutic moieties are provide wherein thechemotherapeutic moiety may be any suitable chemotherapeutic moiety ofchemotherapeutic drugs known in the art that retains cytotoxic activitywhen bonded through a spacing moiety, e.g., an aryl ring and a linker L,to a β-amino acid derivative, β-amino acid analog, or β-amino acidcarboxylic acid (bio)isostere as a LAT1 recognition element provided bythe present disclosure. The conjugate or fusion product of thechemotherapeutic moiety with the β-amino acid derivative, β-amino acidanalog, or β-amino acid carboxylic acid (bio)isostere is simultaneous aselective substrate for the LAT1/4F2hc transporter.

In a third aspect, chemotherapeutic moieties are provided, where in thechemotherapeutic moiety is selected from a nitrogen mustard—N(—CR₂—CR₂—X)₂, a N-monoalkyl or N,N-dialkyl triazene (—N═N—NR₂), ahaloacetamide (—NR—CO—CH₂—X), an epoxide (—CROCR—R), an aziridine(—NC₂H₄), a Michael acceptor (—CR═CR-EWG-), a sulfonate or abissulfonate ester (—OSO₂R or ROSO₂—), an N-nitrosourea (—NR—CO—N(NO)R),a bissulfonyl hydrazine (R″SO₂—NR—N(—)—SO₂R′″, —SO₂—NR—NR′—SO₂R′″, orR″SO₂—NR—NR′—SO₂—), a phosphoramidate (—O—P(═O)(N(R)—CH₂—CH₂—X)₂ or—O—P(═O)(N(—CH₂—CH₂—X)₂)₂, and a radionuclide such as, for example,131-iodine (¹³¹[I]—) or 211-astatine (²¹¹[At]—).

In a fourth aspect, chemotherapeutic moieties of Formula (2) areprovided:

wherein,

A is selected from a bond (“—”), oxygen (—O—), sulfur (—S—), amino(—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenthiocarbonyl (—CH₂—C(═S)—);

Z is selected from a bond (“—”) and oxygen (—O—);

Q is selected from —O⁻ (a negatively charged oxygen atom) that is boundto a positively charged nitrogen atom) and a free electron pair (:),with the proviso that when Q is —O⁻ (a negatively charged oxygen atomthat is bound to a positively charged nitrogen atom), A is selected froma bond (“—”) and methylene (—CH₂—), Z is a bond (“—”), and thechemotherapeutic moiety of Formula (2) is an N-oxide(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂);

each R¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃alkyl; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

In a fifth aspect, pharmaceutical compositions comprising the compoundof claim 1 and a pharmaceutically acceptable vehicle are provided.

In a sixth aspect, uses of compound s of Formula (1) for treating cancerin a patient are provided, comprising administering to a patient in needof such treatment a therapeutically effective amount of the compound ofclaim 1.

DETAILED DESCRIPTION Definitions

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a moiety or substituent. For example,—CONH₂ is attached through the carbon atom.

“Alkyl” refers to a saturated or unsaturated, branched, orstraight-chain, monovalent hydrocarbon radical derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkene,or alkyne. Examples of alkyl groups include methyl; ethyls such asethanyl, ethenyl, and ethynyl; propyls such as propan-1-yl, propan-2-yl,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, but-1-en-1-yl,but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, but-1-yn-1-yl, but-1-yn-3-yl,but-3-yn-1-yl, etc.; and the like. The term “alkyl” is specificallyintended to include groups having any degree or level of saturation,i.e., groups having exclusively carbon-carbon single bonds, groupshaving one or more carbon-carbon double bonds, groups having one or morecarbon-carbon triple bonds, and groups having combinations ofcarbon-carbon single, double, and triple bonds. Where a specific levelof saturation is intended, the terms alkanyl, alkenyl, and alkynyl areused. In certain embodiments, an alkyl group is C₁₋₆ alkyl, C₁₋₅ alkyl,C₁₋₄ alkyl, C₁₋₃ alkyl, and in certain embodiments, ethyl or methyl.

“Alkylsulfanyl” also referred to as “alkylthio”, refers to a radical —SRwhere R is alkyl or cycloalkyl as defined herein. Examples ofalkylsulfanyl groups include methylsulfanyl, ethylsulfanyl,propylsulfanyl, isopropylsulfanyl, butylsulfanyl, andcyclohexylsulfanyl. In certain embodiments, an alkylsulfanyl group isC₁₋₆ alkylsulfanyl, in certain embodiments, C₁₋₅ alkylsulfanyl, incertain embodiments, C₁₋₄ alkylsulfanyl, in certain embodiments, C₁₋₃alkylsulfanyl, in certain embodiments, ethylsulfanyl (ethylthio), and incertain embodiments, methylsulfanyl (methylthio).

“Alkylsulfinyl” refers to a radical —S(O)R where R is alkyl orcycloalkyl as defined herein. Examples of alkylsulfinyl groups includemethylsulfinyl, ethylsulfinyl, propylsulfinyl, isopropylsulfinyl,butylsulfinyl, and cyclohexylsulfinyl. In certain embodiments, analkylsulfinyl group is C₁₋₆ alkylsulfinyl, in certain embodiments, C₁₋₅alkylsulfinyl, in certain embodiments, C₁₋₄ alkylsulfinyl, in certainembodiments, C₁₋₃ alkylsulfinyl, in certain embodiments, ethylsulfinyl,and in certain embodiments, methylsulfinyl.

“Alkylsulfonyl” refers to a radical —S(O)₂R where R is alkyl orcycloalkyl as defined herein. Examples of alkylsulfonyl groups includemethylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl,butylsulfonyl, and cyclohexylsulfonyl. In certain embodiments, analkylsulfonyl group is C₁₋₆ alkylsulfonyl, in certain embodiments, C₁₋₅alkylsulfonyl, in certain embodiments, C₁₋₄ alkylsulfonyl, in certainembodiments, C₁₋₃ alkylsulfonyl, in certain embodiments, ethylsulfonyl,and in certain embodiments, methylsulfonyl.

“Alkoxy” refers to a radical —OR where R is alkyl as defined herein.Examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy.In certain embodiments, an alkoxy group is C₁₋₆ alkoxy, in certainembodiments, C₁₋₅ alkoxy, in certain embodiments, C₁₋₄ alkoxy, incertain embodiments, C₁₋₃ alkoxy, and in certain embodiments, ethoxy ormethoxy.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem. Aryl encompasses 5- and 6-membered carbocyclic aromatic rings,for example, benzene; bicyclic ring systems wherein at least one ring iscarbocyclic and aromatic, for example, naphthalene, indane, andtetralin; and tricyclic ring systems wherein at least one ring iscarbocyclic and aromatic, for example, fluorene. Aryl encompassesmultiple ring systems having at least one carbocyclic aromatic ringfused to at least one carbocyclic aromatic ring, cycloalkyl ring, orheterocycloalkyl ring. For example, aryl includes a phenyl ring fused toa 5- to 7-membered heterocycloalkyl ring containing one or moreheteroatoms selected from N, O, and S. For such fused, bicyclic ringsystems wherein only one of the rings is a carbocyclic aromatic ring,the radical carbon atom may be at the carbocyclic aromatic ring or atthe heterocycloalkyl ring. Examples of aryl groups include groupsderived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like. In certain embodiments, an aryl group isC₆₋₁₀ aryl, C₆₋₉ aryl, C₆₋₈ aryl, and in certain embodiments, phenyl.Aryl, however, does not encompass or overlap in any way with heteroaryl,separately defined herein.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom is replaced with an aryl group.Examples of arylalkyl groups include benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylalkenyl, or arylalkynyl is used. In certainembodiments, an arylalkyl group is C₇₋₁₆ arylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety of the arylalkyl group is C₁₋₆ and the arylmoiety is C₆₋₁₀, in certain embodiments, an arylalkyl group is C₇₋₁₆arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkylgroup is C₁₋₆ and the aryl moiety is C₆₋₁₀. In certain embodiments, anarylalkyl group is C₇₋₉ arylalkyl, wherein the alkyl moiety is C₁₋₃alkyl and the aryl moiety is phenyl. In certain embodiments, anarylalkyl group is C₇₋₁₆ arylalkyl, C₇₋₁₄ arylalkyl, C₇₋₁₂ arylalkyl,C₇₋₁₀ arylalkyl, C₇₋₈ arylalkyl, and in certain embodiments, benzyl.

Bioisosteres are atoms or molecules that fit the broadest definition forisosteres. The concept of bioisosterism is based on the notion thatsingle atom, groups, moieties, or whole molecules, which have chemicaland physical similarities produce similar biological effects. Abioisostere of a parent compound can still be recognized and accepted byits appropriate target, but its functions will be altered as compared tothe parent molecule. Parameters affected with bioisosteric replacementsinclude, for example, size, conformation, inductive and mesomericeffects, polarizability, capacity for electrostatic interactions, chargedistribution, H-bond formation capacity, pKa (acidity), solubility,hydrophobicity, lipophilicity, hydrophilicity, polarity, potency,selectivity, reactivity, or chemical and metabolic stability, ADME(absorption, distribution, metabolism, and excretion). Although commonin pharmaceuticals, carboxyl groups or carboxylic acid functional groups(—CO₂H) in a parent molecule may be replaced with a suitable surrogateor (bio)isostere to overcome chemical or biological shortcomings whileretaining the desired attributes of the parent molecule bearing one ormore carboxyl groups or carboxylic acid functional groups (—CO₂H).Examples of suitable surrogates or (bio)isosteres of carboxyl groups orcarboxylic acid functional groups (—CO₂H) include hydroxamic acids(—CONR¹²OH); boronic acids (—B(OH)(OR¹²), phosphinic acids orderivatives thereof (—PO(OH)R¹²), phosphonic acid or derivatives thereof(—PO(OH)(OR¹²), sulfinic acid (—SOOH), sulfonic acid (—SO₂OH),sulfonamide (—SO₂NHR¹² or —NHSO₂R¹²), sulfonimide or acyl sulfonimide(—SO₂NHCOR¹² or —CONHSO₂R¹²), sulfonylureas (—SO₂NHCONHR¹² or—NHCONHSO₂R¹²), amide (—CONHR¹² or —NHCOR¹²), wherein R¹² in any of theforegoing is selected from hydrogen, C₁₋₆ alkyl, C₁₋₄ fluoroalkyl, C₃₋₆cycloalkyl, and C₆₋₁₀ aryl, acylcyanamide (—CONHCN);2,2,2-trifluoroethan-1-ols (—CH(CF₃)OH), 2,2,2-trifluoromethyl ketonesand hydrates thereof (—COCF₃ and —C(OH)₂CF₃), acidic heterocycles andtheir annular tautomers such as, for example, tetrazole,5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole,5-thioxo-1,2,4-oxadiazole, thiazolidinedione, oxazolidinedione,oxadiazolidinedione, 3-hydroxyisoxazole, 3-hydroxyisothiazole,1-hydroxy-imidazole, 1-hydroxy-pyrazole, 1-hydroxy-triazole,1H-imidazol-2-ol, tetrazole-5-thiol, 3-hydroxyquinolin-2-ones,4-hydroxyquinolin-2-ones, tetronic acid, tetramic acid, mercaptoazolessuch as sulfanyl-1H-imidazole, sulfinyl-1H-imidazole,sulfonyl-1H-imidazole, sulfanyl-1H-triazole, sulfinyl-1H-triazole,sulfonyl-1H-triazole, sulfanyl-1H-1,2,4-triazole,sulfinyl-1H-1,2,4-triazole, sulfonyl-1H-1,2,4-triazole,sulfanyl-1,4-dihydro-1,2,4-triazol-5-one,sulfinyl-1,4-dihydro-1,2,4-triazol-5-one,sulfonyl-1,4-dihydro-1,2,4-triazol-5-one, sulfanyl 1H-tetrazole,sulfanyl 2H-tetrazole, sulfinyl 1H-tetrazole, sulfinyl 2H-tetrazole,sulfonyl 1H-tetrazole, sulfonyl 2H-tetrazole, or sulfonimidamides; and;acidic oxocarbocycles or cyclic polyones and their resonance forms suchas, for example, cyclopentane-1,3-diones, squaric acids, squareamides,mixed squaramates, or 2,6-difluorophenols.

“Compounds” of Formula (1) and moieties of Formula (2) disclosed hereininclude any specific compounds within these formulae. Compounds may beidentified either by their chemical structure and/or chemical name.Compounds are named using the ChemDraw Ultra 12.0 (CambridgeSoft,Cambridge, Mass.) nomenclature program. When the chemical structure andchemical name conflict the chemical structure is determinative of theidentity of the compound. The compounds described herein may compriseone or more stereogenic centers and/or double bonds and therefore mayexist as stereoisomers such as double-bond isomers (i.e., geometricisomers), enantiomers, diastereomers, or atropisomers. Accordingly, anychemical structures within the scope of the specification depicted, inwhole or in part, with a relative configuration encompass all possibleenantiomers and stereoisomers of the illustrated compounds including thestereoisomerically pure form (e.g., geometrically pure, enantiomericallypure, or diastereomerically pure) and enantiomeric and stereoisomericmixtures. Enantiomeric and stereoisomeric mixtures may be resolved intotheir component enantiomers or stereoisomers using separation techniquesor chiral synthesis techniques well known to the skilled artisan.

Compounds of Formula (1) and moieties of Formula (2) include opticalisomers of compounds of Formula (1) and moieties of Formula (2),racemates thereof, and other mixtures thereof. In such embodiments, thesingle enantiomers or diastereomers may be obtained by asymmetricsynthesis or by resolution of the racemates. Resolution of the racematesmay be accomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example a chiral high-pressure liquid chromatography (HPLC)column with chiral stationary phases. In addition, compounds of Formula(1) include (Z)- and (E)-forms (or cis- and trans-forms) of compoundswith double bonds either as single geometric isomers or mixturesthereof.

Compounds of Formula (1) and moieties of Formula (2) may also exist inseveral tautomeric forms including the enol form, the keto form, andmixtures thereof. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.Compounds may exist in unsolvated forms as well as solvated forms,including hydrated forms. Certain compounds may exist in multiplecrystalline, co-crystalline, or amorphous forms. Compounds of Formula(1) include pharmaceutically acceptable salts thereof, orpharmaceutically acceptable solvates of the free acid form of any of theforegoing, as well as crystalline forms of any of the foregoing

Compounds of Formula (1) are also referred to herein as β-substitutedβ-amino acid derivatives and/or as β-substituted β-amino acid analogs.

“Chemotherapeutic moiety” refers to a moiety effective in treatingcancer including, any of those disclosed herein. In certain embodiments,a chemotherapeutic moiety may be any suitable chemotherapeutic moiety ofachemotherapeutic drugs known in the art that retains cytotoxic activitywhen bonded either directly or indirectly through a suitable spacingmoiety to a β-amino acid derivative, β-amino acid analog, or β-aminoacid carboxylic acid (bio)isostere as a LAT1 recognition elementprovided by the present disclosure. The conjugate or fusion product ofthe chemotherapeutic moiety with the β-amino acid derivative, β-aminoacid analog, or β-amino acid carboxylic acid (bio)isostere issimultaneous a selective substrate for the LAT1/4F2hc transporter.

In certain embodiments, the chemotherapeutic moiety, is selected from anitrogen mustard (—N(—CR₂—CR₂—X)₂), a N-monoalkyl or N,N-dialkyltriazene (—N═N—NR₂), a haloacetamide (—NR—CO—CH₂—X), an epoxide(—CROCR—R), an aziridine (—NC₂H₄), a Michael acceptor (—CR═CR-EWG-), asulfonate or a bissulfonate ester (—OSO₂R or ROSO₂—), an N-nitrosourea(—NR—CO—N(NO)R), a bissulfonyl hydrazine (R″SO₂—NR—N(—)—SO₂R′″,—SO₂—NR—NR′—SO₂R′″, or R″SO₂—NR—NR′—SO₂—), a phosphoramidate(—O—P(═O)(N(R)—CH₂—CH₂—X)₂ or —O—P(═O)(N(—CH₂—CH₂—X)₂)₂, and aradionuclide such as, for example, 131-iodine (¹³¹[I]—) or 211-astatine(²¹¹[At]—).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety is a moiety Formula (2a):-A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)  (2a)wherein,

A is selected from a bond (“—”), oxygen (—O—), sulfur (—S—), amino(—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenthiocarbonyl (—CH₂—C(═S)—);

Z is selected from a bond (“—”) and oxygen (—O—);

Q is selected from —O— (a negatively charged oxygen atom) that is boundto a positively charged nitrogen atom) and a free electron pair (:),with the proviso that when Q is —O— (a negatively charged oxygen atomthat is bound to a positively charged nitrogen atom), A is selected froma bond (“—”) and methylene (—CH₂—), Z is a bond (“—”), and thechemotherapeutic moiety of Formula (2) is an N-oxide(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂); and

each R¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃alkyl; and

-   -   each R⁹ is independently selected from fluoro (—F), chloro        (—Cl), bromo (—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰,        wherein R⁴⁰ is selected from C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl        sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄        (per)fluoroalkyl), and (substituted) aryl sulfonate (—OSO₂R⁴⁰,        wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

“Cycloalkyl” refers to a saturated or partially unsaturated cyclic alkylradical. In certain embodiments, a cycloalkyl group is C₃₋₆ cycloalkyl,C₃₋₅ cycloalkyl, C₅₋₆ cycloalkyl, cyclopropyl, cyclopentyl, and incertain embodiments, cyclohexyl. In certain embodiments, cycloalkyl isselected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Cycloalkylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom is replaced with a cycloalkylgroup as defined herein. Where specific alkyl moieties are intended, thenomenclature cycloalkylalkyl, cycloalkylalkenyl, or cycloalkylalkynyl isused. In certain embodiments, a cycloalkylalkyl group is C₄₋₃₀cycloalkylalkyl, e.g., the alkanyl, alkenyl, or alkynyl moiety of thecycloalkylalkyl group is C₁₋₁₀ and the cycloalkyl moiety of thecycloalkylalkyl moiety is C₃₋₂₀, and in certain embodiments, ancycloalkylalkyl group is C₄₋₂₀ cycloalkylalkyl, e.g., the alkanyl,alkenyl, or alkynyl moiety of the cycloalkylalkyl group is C₁₋₈ and thecycloalkyl moiety of the cycloalkylalkyl group is C₃₋₁₂. In certainembodiments, cycloalkylalkyl is C₄₋₉ cycloalkylalkyl, wherein the alkylmoiety of the cycloalkylalkyl group is C₁₋₃ alkyl, and the cycloalkylmoiety of the cycloalkylalkyl group is C₃₋₆ cycloalkyl. In certainembodiments, a cycloalkylalkyl group is C₄₋₁₂ cycloalkylalkyl, C₄₋₁₀cycloalkylalkyl, C₄₋₈ cycloalkylalkyl, and C₄₋₆ cycloalkylalkyl. Incertain embodiments a cycloalkylalkyl group is cyclopropylmethyl(—CH₂-cyclo-C₃H₅), cyclopentylmethyl (—CH₂-cyclo-C₅H₉), orcyclohexylmethyl (—CH₂-cyclo-C₆H₁₁). In certain embodiments acycloalkylalkyl group is cyclopropylethenyl (—CH═CH-cyclo-C₃H₅),cyclopentylethynyl (—C≡C-cyclo-C₅H₉), or the like.

“Cycloalkylheteroalkyl” by itself or as part of another substituentrefers to a heteroalkyl group in which one or more of the carbon atoms(and certain associated hydrogen atoms) of an alkyl group areindependently replaced with the same or different heteroatomic group orgroups and in which one of the hydrogen atoms bonded to a carbon atom isreplaced with a cycloalkyl group. Where specific alkyl moieties areintended, the nomenclature cycloalkylheteroalkanyl,cycloalkylheteroalkenyl, and cycloalkylheteroalkynyl is used. In certainembodiments of cycloalkylheteroalkyl, the heteroatomic group is selectedfrom —O—, —S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments,the heteroatomic group is selected from —O— and —NH—, and in certainembodiments the heteroatomic group is —O— or —NH—.

“Cycloalkyloxy” refers to a radical —OR where R is cycloalkyl as definedherein. Examples of cycloalkyloxy groups include cyclopropyloxy,cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. In certainembodiments, a cycloalkyloxy group is C₃₋₆ cycloalkyloxy, in certainembodiments, C₃₋₅ cycloalkyloxy, in certain embodiments, C₅₋₆cycloalkyloxy, and in certain embodiments, cyclopropyloxy,cyclobutyloxy, cyclopentyloxy, or cyclohexyloxy.

“Disease” refers to a disease, disorder, condition, or symptom of any ofthe foregoing.

“Fluoroalkyl” refers to an alkyl group as defined herein in which one ormore of the hydrogen atoms is replaced with a fluoro. In certainembodiments, a fluoroalkyl group is C₁₋₆ fluoroalkyl, C₁₋₅ fluoroalkyl,C₁₋₄ fluoroalkyl, and C₁₋₃ fluoroalkyl. In certain embodiments, thefluoroalkyl group is pentafluoroethyl (—CF₂CF₃), and in certainembodiments, trifluoromethyl (—CF₃).

“Fluoroalkoxy” refers to an alkoxy group as defined herein in which oneor more of the hydrogen atoms is replaced with a fluoro. In certainembodiments, a fluoroalkoxy group is C₁₋₆ fluoroalkoxy, C₁₋₅fluoroalkoxy, C₁₋₄ fluoroalkoxy C₁₋₃, or fluoroalkoxy, and in certainembodiments, —OCF₂CF₃ or —OCF₃.

“β-Substituted β-amino acid derivative” refers to β-substituted β-aminoacid derivatives having a carboxyl group, e.g., β-substituted β-aminoacid.

“β-Substituted β-amino acid analog” refers to β-substituted β-amino acidderivatives in which the carboxyl group is replaced with a phosphinicacid group, a sulfinic acid group, or others, e.g.,β-aminopropylphosphinic acids, β-aminopropylsulfinic acids, and others.

“Halogen” refers to a fluoro, chloro, bromo, or iodo group.

“Heteroalkoxy” refers to an alkoxy group in which one or more of thecarbon atoms are replaced with a heteroatom. In certain embodiments, theheteroalkoxy group is C₁₋₆ heteroalkoxy, in certain embodiments, C₁₋₅heteroalkoxy, in certain embodiments, C₁₋₄ heteroalkoxy, and in certainembodiments, C₁₋₃ heteroalkoxy. In certain embodiments of heteroalkoxy,the heteroatomic group is selected from —O—, —S—, —NH—, —NR—, —SO₂—, and—SO₂—, in certain embodiments, the heteroatomic group is selected from—O— and —NH—, and in certain embodiments the heteroatomic group is —O—and —NH—. In certain embodiments, a heteroalkoxy group is C₁₋₆heteroalkoxy, C₁₋₅ heteroalkoxy, C₁₋₄ heteroalkoxy, and in certainembodiments C₁₋₃ heteroalkoxy.

“Heteroalkyl” by itself or as part of another substituent refer to analkyl group in which one or more of the carbon atoms (and certainassociated hydrogen atoms) are independently replaced with the same ordifferent heteroatomic group or groups. Examples of heteroatomic groupsinclude —O—, —S—, —NH—, —NR—, —O—O—, —S—S—, ═N—N═, —N═N—, —N═N—NR—,—PR—, —P(O)OR—, —P(O)R—, —POR—, —SO—, —SO₂—, —Sn(R)₂—, and the like,where each R is independently selected from hydrogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₆₋₁₂ aryl, substituted C₆₋₁₂ aryl, C₇₋₁₈arylalkyl, substituted C₇₋₁₈ arylalkyl, C₃₋₇ cycloalkyl, substitutedC₃₋₇ cycloalkyl, C₃₋₇ heterocycloalkyl, substituted C₃₋₇heterocycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₆₋₁₂heteroaryl, substituted C₆₋₁₂ heteroaryl, C₇₋₁₈ heteroarylalkyl, andsubstituted C₇₋₁₈ heteroarylalkyl. In certain embodiments, each R isindependently selected from hydrogen and C₁₋₃ alkyl. Reference to, forexample, a C₁₋₆ heteroalkyl, means a C₁₋₆ alkyl group in which at leastone of the carbon atoms (and certain associated hydrogen atoms) isreplaced with a heteroatom. For example, C₁₋₆ heteroalkyl includesgroups having five carbon atoms and one heteroatom, groups having fourcarbon atoms and two heteroatoms, etc. In certain embodiments ofheteroalkyl, the heteroatomic group is selected from —O—, —S—, —NH—,—N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments, the heteroatomicgroup is selected from —O— and —NH—, and in certain embodiments, theheteroatomic group is —O— or —NH—. In certain embodiments, a heteroalkylgroup is C₁₋₆ heteroalkyl, C₁₋₅ heteroalkyl, or C₁₋₄ heteroalkyl, and incertain embodiments, C₁₋₃ heteroalkyl.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring system.Heteroaryl encompasses multiple ring systems having at least oneheteroaromatic ring fused to at least one other ring, which may bearomatic or non-aromatic. For example, heteroaryl encompasses bicyclicrings in which one ring is heteroaromatic and the second ring is aheterocycloalkyl ring. For such fused, bicyclic heteroaryl ring systemswherein only one of the rings contains one or more heteroatoms, theradical carbon may be at the aromatic ring or at the heterocycloalkylring. In certain embodiments, when the total number of N, S, and O atomsin the heteroaryl group exceeds one, the heteroatoms may or may not beadjacent to one another. In certain embodiments, the total number ofheteroatoms in the heteroaryl group is not more than two. In certainembodiments of heteroaryl, the heteroatomic group is selected from —O—,—S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments, theheteroatomic group is selected from —O— and —NH—, and in certainembodiments the heteroatomic group is —O— or —NH—. In certainembodiments, a heteroaryl group is selected from C₅₋₁₀ heteroaryl, C₅₋₉heteroaryl, C₅₋₈ heteroaryl, C₅₋₇ heteroaryl, C₅₋₆ heteroaryl, and incertain embodiments, is C₅ heteroaryl and C₆ heteroaryl.

Examples of heteroaryl groups include groups derived from acridine,arsindole, carbazole, α-carboline, chromane, chromene, cinnoline, furan,imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,thiazolidine, oxazolidine, and the like. In certain embodiments,heteroaryl groups are those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole, or pyrazine. For example, in certain embodiments, heteroaryl isC₅ heteroaryl and is selected from furyl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, isothiazolyl, or isoxazolyl. In certain embodiments,heteroaryl is C₆ heteroaryl, and is selected from pyridinyl, pyrazinyl,pyrimidinyl, and pyridazinyl.

“Heteroarylalkyl” refers to an arylalkyl group in which one of thecarbon atoms (and certain associated hydrogen atoms) is replaced with aheteroatom. In certain embodiments, a heteroarylalkyl group is C₆₋₁₆heteroarylalkyl, C₆₋₁₄ heteroarylalkyl, C₆₋₁₂ heteroarylalkyl, C₆₋₁₀heteroarylalkyl, C₆₋₈ heteroarylalkyl, or C₇ heteroarylalkyl, and incertain embodiments, C₆ heteroarylalkyl. In certain embodiments ofheteroarylalkyl, the heteroatomic group is selected from —O—, —S—, —NH—,—N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments, the heteroatomicgroup is selected from —O— and —NH—, and in certain embodiments theheteroatomic group is —O— or —NH—.

“Heterocycloalkyl” by itself or as part of another substituent refers toa saturated or unsaturated cyclic alkyl radical in which one or morecarbon atoms (and certain associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom; or to a parent aromaticring system in which one or more carbon atoms (and certain associatedhydrogen atoms) are independently replaced with the same or differentheteroatom such that the ring system violates the Hückel-rule. Examplesof heteroatoms to replace the carbon atom(s) include N, P, O, S, and Si.Examples of heterocycloalkyl groups include groups derived fromepoxides, azirines, thiiranes, imidazolidine, morpholine, piperazine,piperidine, pyrazolidine, pyrrolidine, quinuclidine, and the like. Incertain embodiments, heterocycloalkyl is C₅ heterocycloalkyl and isselected from pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl,imidazolidinyl, oxazolidinyl, thiazolidinyl, doxolanyl, and dithiolanyl.In certain embodiments, heterocycloalkyl is C₆ heterocycloalkyl and isselected from piperidinyl, tetrahydropyranyl, piperizinyl, oxazinyl,dithianyl, and dioxanyl. In certain embodiments a heterocycloalkyl groupis C₃₋₆ heterocycloalkyl, C₃₋₅ heterocycloalkyl, C₅₋₆ heterocycloalkyl,and in certain embodiments, C₅ heterocycloalkyl or C₆ heterocycloalkyl.In certain embodiments of heterocycloalkyl, the heteroatomic group isselected from —O—, —S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certainembodiments, the heteroatomic group is selected from —O— and —NH—, andin certain embodiments the heteroatomic group is —O— or —NH—.

“Heterocycloalkylalkyl” refers to a cycloalkylalkyl group in which oneor more carbon atoms (and certain associated hydrogen atoms) of thecycloalkyl ring are independently replaced with the same or differentheteroatom. In certain embodiments, the heterocycloalkylalkyl is C₄₋₁₂heterocycloalkylalkyl, C₄₋₁₀ heterocycloalkylalkyl, C₄₋₈heterocycloalkylalkyl, C₄₋₆ heterocycloalkylalkyl, or C₆₋₇heterocycloalkylalkyl, and in certain embodiments, C₆heterocycloalkylalkyl or C₇ heterocycloalkylalkyl. In certainembodiments of heterocycloalkylalkyl, the heteroatomic group is selectedfrom —O—, —S—, —NH—, —N(—CH₃)—, —SO—, and —SO₂—, in certain embodiments,the heteroatomic group is selected from —O— and —NH—, and in certainembodiments, the heteroatomic group is —O— or —NH—.

“Mesyl” refers to the group —OS(O)₂Me or —OMs.

“Parent aromatic ring system” refers to an unsaturated cyclic orpolycyclic ring system having a cyclic conjugated π (pi) electron systemwith 4n+2 electrons (Hückel rule). Included within the definition of“parent aromatic ring system” are fused ring systems in which one ormore of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Examples of parent aromatic ring systems includeaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Parent heteroaromatic ring system” refers to an aromatic ring system inwhich one or more carbon atoms (and any associated hydrogen atoms) areindependently replaced with the same or different heteroatom in such away as to maintain the continuous π-electron system characteristic ofaromatic systems and a number of π-electrons corresponding to the Hückelrule (4n+2). Examples of heteroatoms to replace the carbon atoms includeN, P, O, S, and Si, etc. Specifically included within the definition of“parent heteroaromatic ring systems” are fused ring systems in which oneor more of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, arsindole, benzodioxan,benzofuran, chromane, chromene, indole, indoline, xanthene, etc.Examples of parent heteroaromatic ring systems include arsindole,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,thiazolidine, oxazolidine, and the like.

“Patient” refers to a mammal, for example, a human. The term “patient”is used interchangeably with “subject.”

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the parent compound.Such salts include acid addition salts, formed with inorganic acids andone or more protonable functional groups such as primary, secondary, ortertiary amines within the parent compound. Examples of inorganic acidsinclude hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. In certain embodiments the salts areformed with organic acids such as acetic acid, propionic acid, hexanoicacid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lacticacid, malonic acid, succinic acid, malic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, lauryl sulfuric acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, andthe like. In certain embodiments, a salt is formed when one or moreacidic protons present in the parent compound are replaced by a metalion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminumion, or combinations thereof; or coordinates with an organic base suchas ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, andthe like. In certain embodiments, a pharmaceutically acceptable salt isthe hydrochloride salt. In certain embodiments, a pharmaceuticallyacceptable salt is the sodium salt. In certain embodiments wherein acompound has two or more ionizable groups, a pharmaceutically acceptablesalt comprises one or more counterions, such as a bi-salt, for example,a dihydrochloride salt.

The term “pharmaceutically acceptable salt” includes hydrates and othersolvates, as well as salts in crystalline or non-crystalline form. Wherea particular pharmaceutically acceptable salt is disclosed, it isunderstood that the particular salt (e.g., a hydrochloride salt) is anexample of a salt, and that other salts may be formed using techniquesknown to one of skill in the art. Additionally, one of skill in the artwould be able to convert the pharmaceutically acceptable salt to thecorresponding compound, free base and/or free acid, using techniquesgenerally known in the art. See also: Stahl and Wermuth, C. G.(Editors), Handbook of Pharmaceutical Salts, Wiley-VCH, Weinheim,Germany, 2008.

“Pharmaceutically acceptable vehicle” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable excipient, a pharmaceutically acceptablecarrier, or a combination of any of the foregoing with which a compoundprovided by the present disclosure may be administered to a patient andwhich does not destroy the pharmacological activity thereof and which isnon-toxic when administered in doses sufficient to provide atherapeutically effective amount of the compound.

“Pharmaceutical composition” refers to a compound of Formula (1) or apharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable vehicle, with which the compound of Formula(1) or a pharmaceutically acceptable salt thereof is administered to apatient. Pharmaceutically acceptable vehicles are known in the art.

“Solvate” refers to a molecular complex of a compound with one or moresolvent molecules in a stoichiometric or non-stoichiometric amount. Suchsolvent molecules are those commonly used in the pharmaceutical arts,which are known to be innocuous to a patient, e.g., water, ethanol, andthe like. A molecular complex of a compound or moiety of a compound anda solvent can be stabilized by non-covalent intra-molecular forces suchas, for example, electrostatic forces, van der Waals forces, or hydrogenbonds. The term “hydrate” refers to a solvate in which the one or moresolvent molecules is water.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s). Incertain embodiments, each substituent is independently selected fromhalogen, —OH, —CN, —CF₃, —OCF₃, ═O, —NO₂, C₁₋₆ alkoxy, C₁₋₆ alkyl,—COOR, —NR₂, and —CONR₂; wherein each R is independently selected fromhydrogen and C₁₋₆ alkyl. In certain embodiments, each substituent isindependently selected from halogen, —NH₂, —OH, C₁₋₃ alkoxy, and C₁₋₃alkyl, trifluoromethoxy, and trifluoromethyl. In certain embodiments,each substituent is independently selected from —OH, methyl, ethyl,trifluoromethyl, methoxy, ethoxy, and trifluoromethoxy. In certainembodiments, each substituent is selected from C₁₋₃ alkyl, ═O, C₁₋₃alkyl, C₁₋₃ alkoxy, and phenyl. In certain embodiments, each substituentis selected from —OH, —NH₂, C₁₋₃ alkyl, and C₁₋₃ alkoxy.

“Treating” or “treatment” of a disease refers to arresting orameliorating a disease or at least one of the clinical symptoms of adisease or disorder, reducing the risk of acquiring a disease or atleast one of the clinical symptoms of a disease, reducing thedevelopment of a disease or at least one of the clinical symptoms of thedisease or reducing the risk of developing a disease or at least one ofthe clinical symptoms of a disease. “Treating” or “treatment” alsorefers to inhibiting the disease, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both, and to inhibiting atleast one physical parameter or manifestation that may or may not bediscernible to the patient. In certain embodiments, “treating” or“treatment” refers to delaying the onset of the disease or at least oneor more symptoms thereof in a patient who may be exposed to orpredisposed to a disease or disorder even though that patient does notyet experience or display symptoms of the disease.

“Therapeutically effective amount” refers to the amount of a compoundthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease, is sufficient to affect suchtreatment of the disease or symptom thereof. The “therapeuticallyeffective amount” may vary depending, for example, on the compound, thedisease and/or symptoms of the disease, severity of the disease and/orsymptoms of the disease or disorder, the age, weight, and/or health ofthe patient to be treated, and the judgment of the prescribingphysician. An appropriate amount in any given instance may beascertained by those skilled in the art or capable of determination byroutine experimentation.

“Therapeutically effective dose” refers to a dose that provideseffective treatment of a disease or disorder in a patient. Atherapeutically effective dose may vary from compound to compound, andfrom patient to patient, and may depend upon factors such as thecondition of the patient and the route of delivery. A therapeuticallyeffective dose may be determined in accordance with routinepharmacological procedures known to those skilled in the art.

“Triflyl” refers to the group —OS(O)₂CF₃ or —OTf.

Reference is now made in detail to certain embodiments of compounds,compositions, and methods. The disclosed embodiments are not intended tobe limiting of the claims. To the contrary, the claims are intended tocover all alternatives, modifications, and equivalents.

LAT1/4F2hc Transporter

The GenBank accession number for human LAT1/4F2hc isNP_003477/NP_002385. Unless otherwise apparent from the context,reference to a transporter such as LAT1/4F2hc (as well as othertransporters disclosed herein) includes the amino acid sequencedescribed in or encoded by the GenBank reference number, and, allelic,cognate and induced variants and fragments thereof retaining essentiallythe same transporter activity. Usually such variants show at least 90%sequence identity to the exemplary Genbank nucleic acid or amino acidsequence. Allelic variants at the DNA level are the result of geneticvariation between individuals of the same species. Some allelic variantsat the DNA level that cause substitution, deletion or insertion of aminoacids in proteins encoded by the DNA result in corresponding allelicvariation at the protein level. Cognate forms of a gene refer tovariation between structurally and functionally related genes betweenspecies. For example, the human gene showing the greatest sequenceidentity and closest functional relationship to a mouse gene is thehuman cognate form of the mouse gene.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm enables calculation of the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters. Optimal alignment ofsequences for comparison may be conducted by methods known to thoseskilled in the art.

Compounds

In certain embodiments, anti-cancer agents provided by the presentdisclosure are compounds of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

at least one of R¹ and R⁵ is independently selected from halogen,—N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(OR¹⁰)(R¹⁰), —NO₂, —NO, —N(R¹⁰)(S(═O)R¹⁰),—N(R¹⁰)(S(═O)₂R¹⁰), —N(R¹⁰)(C(O)R¹⁰), —N(R¹⁰)(C(O)OR),—N(R¹⁰)(C(O)N(R¹⁰)₂, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, —SH, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, —S(O)N(R¹⁰)₂,—S(O)₂N(R¹⁰)₂, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₃₋₆cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy, substitutedC₃₋₆ cycloalkyloxy, C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂cycloalkylalkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl,substituted C₇₋₁₆ arylalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ heteroalkoxy, substituted C₁₋₆ heteroalkoxy, C₃₋₆heterocycloalkyl, substituted C₃₋₆ heterocycloalkyl, C₄₋₁₂heterocycloalkylalkyl, substituted C₄₋₁₂ heterocycloalkylalkyl, C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₆₋₁₆ heteroarylalkyl, andsubstituted C₆₋₁₆ heteroarylalkyl;

one of R¹, R², R³, R⁴, and R⁵ comprises a chemotherapeutic moiety;

each of the other of R¹, R², R³, R⁴, and R⁵ is independently selectedfrom hydrogen, deuterio, halogen, —OH, —N(R¹⁰)₂, —NO₂, —NO, —CN,—COOR¹⁰, —CON(R¹⁰)₂, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₃₋₆ cycloalkyl,substituted C₃₋₆ cycloalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy,substituted C₁₋₆ heteroalkoxy, C₄₋₈ cycloalkylalkyl, and C₄₋₈cycloalkylheteroalkyl;

R⁶ is selected from a carboxylic acid (—COOH), a carboxylic acid analog,and a carboxylic acid (bio)isostere;

each R⁷ is independently selected from hydrogen, deuterio, halogen,hydroxyl, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, benzyl, and phenyl; or two R⁷together with the carbon to which they are bonded form a ring selectedfrom a C₃₋₆ cycloalkyl ring and a C₃₋₆ heterocycloalkyl ring;

R⁸ is selected from hydrogen, deuterio, halogen, C₁₋₆ alkyl, substitutedC₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy,substituted C₁₋₆ alkoxy, C₁₋₆ heteroalkoxy, substituted C₁₋₆heteroalkoxy, C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆cycloalkyloxy, substituted C₃₋₆ cycloalkyloxy, —OH, —COOR¹⁰, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₆ cycloalkyl, and phenyl;

each R¹⁰ is independently selected from hydrogen, C₁₋₄ alkyl and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

L is —(X)_(a)—, wherein,

-   -   each X is independently selected from a bond (“—”) and        —C(R¹⁶)₂—, wherein each R¹⁶ is independently selected from        hydrogen, deuterio, halogen, hydroxyl, C₁₋₄ alkyl, and C₁₋₄        alkoxy, or two R¹⁶ together with the carbon to which they are        bonded form a C₃₋₆ cycloalkyl ring or a C₃₋₆ heterocycloalkyl        ring, —O—, —S—, —SO—, —SO₂—, —CO—, and —N(R¹⁷)—, wherein R¹⁷ is        selected from hydrogen, and C₁₋₄ alkyl; and    -   a is selected from 0, 1, 2, 3, and 4; and

each substituent is independently selected from halogen, —OH, —NH₂,—N(R¹⁰)₂, —NO₂, —CF₃, ═O (oxo), C₁₋₃ alkyl, C₁₋₃ alkoxy, and phenyl;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl.

In certain embodiments in compounds of Formula (1), R¹ comprises achemotherapeutic moiety, R² comprises a chemotherapeutic moiety, R³comprises a chemotherapeutic moiety, R⁴ comprises a chemotherapeuticmoiety, and in certain embodiments, R⁵ comprises a chemotherapeuticmoiety.

In certain embodiments of a compound of Formula (1), a chemotherapeuticmoiety may be any suitable chemotherapeutic moiety of a chemotherapeuticdrug known in the art that retains cytotoxic activity when bondedthrough a spacing moiety, e.g., an aryl ring and a linker L, to aβ-amino acid derivative, β-amino acid analog, or β-amino acid carboxylicacid (bio)isostere as a LAT1 recognition element provided by the presentdisclosure. The conjugate or fusion product of the chemotherapeuticmoiety with the β-amino acid derivative, β-amino acid analog, or β-aminoacid carboxylic acid (bio)isostere is simultaneous a selective substratefor the LAT1/4F2hc transporter.

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety, comprises a nitrogen mustard —N(—CR₂—CR₂—X)₂, aN-monoalkyl or N,N-dialkyl triazene (—N═N—NR₂), a haloacetamide(—NR—CO—CH₂—X), an epoxide (—CROCR—R), an aziridine (—NC₂H₄), a Michaelacceptor (—CR═CR-EWG-), a sulfonate or a bissulfonate ester (—OSO₂R orROSO₂—), an N-nitrosourea (—NR—CO—N(NO)R), a bissulfonyl hydrazine(R″SO₂—NR—N(—)—SO₂R′″, —SO₂—NR—NR′—SO₂R′″, or R″SO₂—NR—NR′—SO₂—), aphosphoramidate (—O—P(═O)(N(R)—CH₂—CH₂—X)₂ or —O—P(═O)(N(—CH₂—CH₂—X)₂)₂,and a radionuclide such as, for example, 131-iodine (¹³¹[I]—) or211-astatine (²¹¹[At]—).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety is a moiety Formula (2):

wherein,

A is selected from a bond (“—”), oxygen (—O—), sulfur (—S—), amino(—NR¹⁰—), methylene (—CH₂—), methyleneoxy (—CH₂—O—), oxycarbonyl(—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl (—NR¹⁰—C(═O)—),oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl (—S—C(═S)—),aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl (—CH₂—O—C(═O)—),methylenethiocarbonyl (—CH₂—S—C(═O)—), methyleneaminocarbonyl(—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl (—CH₂—O—C(═S)—),methylenethiothiocarbonyl (—CH₂—S—C(═S)—), methyleneaminothiocarbonyl(—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—), methylencarbonyl (—CH₂—C(═O)—),thiocarbonyl (—C(═S)—), and methylenthiocarbonyl (—CH₂—C(═S)—);

Z is selected from a bond (“—”) and oxygen (—O—);

Q is selected from —O— (a negatively charged oxygen atom) that is boundto a positively charged nitrogen atom) and a free electron pair (:),with the proviso that when Q is —O— (a negatively charged oxygen atomthat is bound to a positively charged nitrogen atom), A is selected froma bond (“—”) and methylene (—CH₂—), Z is a bond (“—”), and thechemotherapeutic moiety of Formula (2) is an N-oxide(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂);

each R¹¹ is independently selected from hydrogen, deuterio, and C₁₋₃alkyl; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

In certain embodiments, a chemotherapeutic moiety of Formula (2) isselected from the structure-A-N(—Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹ and(-A-N⁺(—O⁻)(—C(R¹¹)₂—C(R¹¹)₂—R⁹)₂, wherein,

A is selected from a bond (“—”), methylene (—CH₂—), oxygen (—O—),methyleneoxy (—CH₂—O—), oxycarbonyl (—O—C(═O)—), methyleneoxycarbonyl(—CH₂—O—C(═O)—), carbonyl (—C(═O)—), and methylenecarbonyl(—CH₂—C(═O)—);

each R¹¹ is independently selected from hydrogen and deuterio; and

each R⁹ is independently selected from fluoro (—F), chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, whereinR⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl), and (substituted) arylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₆₋₁₀ aryl).

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹, wherein,

A is a bond (“—”);

Q is a free electron pair (:);

Z is a bond (“—”);

each R¹¹ is independently selected from hydrogen and deuterio; and

each R⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo(—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n are independentlyselected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein,

A is methylene (—CH₂—);

Q is a free electron pair (:);

Z is a bond (“—”);

each R¹¹ is independently selected from hydrogen and deuterio; and

each R⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo(—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is—CH₂—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein A isa bond (“—”), Q is a negatively charged oxygen (—O—), Z is a bond (“—”),each R¹¹ is independently selected from hydrogen and deuterio; and eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), andC₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected fromC₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein A ismethylene (—CH₂—), Q is a negatively charged oxygen (—O—), Z is a bond(“—”), each R¹¹ is independently selected from hydrogen and deuterio;and each R⁹ is independently selected from chloro (—Cl), bromo (—Br),iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is—CH₂—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein A isa bond (“—”), Q is a free electron pair (:), Z is oxygen, each R¹¹ isindependently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl) and the chemotherapeutic moiety is—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein A ismethylene (—CH₂—), Q is a free electron pair (:), Z is oxygen, each R¹¹is independently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl) and the chemotherapeutic moiety is—CH₂—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein A isoxygen (—O—), Q is a free electron pair (:), Z is a bond (“—”), each R¹¹is independently selected from hydrogen and deuterio; and each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I), alkylsulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl) and the chemotherapeutic moiety is—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methyleneoxy (—CH₂—O—), Q is a free electron pair (:), Z is a bond(“—”), each R¹¹ is independently selected from hydrogen and deuterio;and each R⁹ is independently selected from chloro (—Cl), bromo (—Br),iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from and C₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moietyis —CH₂—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais a carbonyl (—CO—), Q is a free electron pair (:), Z is a bond (“—”),each R¹¹ is independently selected from hydrogen and deuterio; and eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), andC₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected fromC₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais methylenecarbonyl (—CH₂—CO—), Q is a free electron pair (:), Z is abond (“—”), each R¹¹ is independently selected from hydrogen anddeuterio; and each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selectedfrom C₁₋₄ alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰,wherein R⁴⁰ is selected from C₁₋₄ (per)fluoroalkyl) and thechemotherapeutic moiety is —CH₂—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂,wherein m and n are independently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais oxycarbonyl (—O—CO—), Q is a free electron pair (:), Z is a bond(“—”), each R¹¹ is independently selected from hydrogen and deuterio;and each R⁹ is independently selected from chloro (—Cl), bromo (—Br),iodo (—I), alkyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄alkyl), and C₁₋₄ (per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ isselected from C₁₋₄ (per)fluoroalkyl) and the chemotherapeutic moiety is—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments, a chemotherapeutic moiety of Formula (2) has thestructure -A-NQ(-Z—C(R¹¹)₂—C(R¹¹)₂—R⁹)(—C(R¹¹)₂—C(R¹¹)₂—R⁹), wherein Ais a methyleneoxycarbonyl (—CH₂—O—CO—), each R¹¹ is independentlyselected from hydrogen and deuterio; and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), alkyl sulfonate(—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄ alkyl), and C₁₋₄(per)fluoroalklyl sulfonate (—OSO₂R⁴⁰, wherein R⁴⁰ is selected from C₁₋₄(per)fluoroalkyl) and the chemotherapeutic moiety is—CH₂—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2.

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂,wherein m and n are independently selected from 0, 1, and 2, and each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—N⁺(—O⁻)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2, and each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—N(—O—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹),wherein m and n are independently selected from 0, 1, and 2, and each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—O—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises—CH₂—O—CO—N(—CH_(2-m)D_(m)-CH_(2-n)D_(n)-R⁹)₂, wherein m and n areindependently selected from 0, 1, and 2, and each R⁹ is independentlyselected from chloro (—Cl), bromo (—Br), iodo (—I), methylsulfonyloxy(—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, wherein eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, whereineach R⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo(—I), methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy(—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),wherein each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), methylsulfonyloxy (—OSO₂CH₃), andtrifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),wherein each R⁹ is independently selected from chloro (—Cl), bromo(—Br), iodo (—I), methylsulfonyloxy (—OSO₂CH₃), andtrifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —O—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—O—N(—CH₂—CH₂—R⁹)₂, wherein eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—CO—N(—CH₂—CH₂—R⁹)₂, wherein eachR⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —O—CO—N(—CH₂—CH₂—R⁹)₂, wherein m and nare independently selected from 0, 1, and 2, and wherein each R⁹ isindependently selected from chloro (—Cl), bromo (—Br), iodo (—I),methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy (—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), thechemotherapeutic moiety comprises —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, whereineach R⁹ is independently selected from chloro (—Cl), bromo (—Br), iodo(—I), methylsulfonyloxy (—OSO₂CH₃), and trifluoromethylsulfonyloxy(—OSO₂CF₃).

In certain embodiments of a compound of Formula (1), R⁶ is selected fromcarboxylic acid (—COOH), hydroxamic acids (—CONR¹²OH), boronic acids(—B(OH)(OR¹²), phosphinic acids or derivatives thereof (—PO(OH)R¹²), andphosphonic acid or derivatives thereof (—PO(OH)(OR¹²)), sulfinic acid(—SOOH), sulfonic acid (—SO₂OH), sulfonamide (—SO₂NHR¹² or —NHSO₂R¹²),sulfonimide or acyl sulfonimide (—SO₂NHCOR¹² or —CONHSO₂R¹²),sulfonylureas (—SO₂NHCONHR¹² or —NHCONHSO₂R¹²), amide (—CONHR¹² or—NHCOR¹²), acylcyanamide (—CONHCN), 2,2,2-trifluoroethan-1-ols(—CH(CF₃)OH), 2,2,2-trifluoromethyl ketones and hydrates thereof (—COCF₃and —C(OH)₂CF₃), acidic heterocycles and annular tautomers of any of theforegoing, and acidic oxocarbocycles or cyclic polyones and resonanceforms of any of the foregoing; wherein R¹² is selected from hydrogen,C₁₋₆ alkyl, C₁₋₄ fluoroalkyl, C₃₋₆ cycloalkyl, and C₆₋₁₀ aryl.

In certain embodiments of a compound of Formula (1), the acidicheterocycle and annular tautomers is selected from 1H-tetrazole,5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole,5-thioxo-1,2,4-oxadiazole, thiazolidinedione, oxazolidinedione,oxadiazolidinedione, 3-hydroxyisoxazole, 3-hydroxyisothiazole,1-hydroxy-imidazole, 1-hydroxy-pyrazole, 1-hydroxy-triazole,1H-imidazol-2-ol, tetrazole-5-thiol, 3-hydroxyquinolin-2-one,4-hydroxyquinolin-2-ones, tetronic acid, tetramic acid, mercaptoazolessuch as sulfanyl-1H-imidazole, sulfinyl-1H-imidazole,sulfonyl-1H-imidazole, sulfanyl-1H-triazole, sulfinyl-1H-triazole,sulfonyl-1H-triazole, sulfanyl-1H-1,2,4-triazole,sulfinyl-1H-1,2,4-triazole, sulfonyl-1H-1,2,4-triazole,sulfanyl-1,4-dihydro-1,2,4-triazol-5-one,sulfinyl-1,4-dihydro-1,2,4-triazol-5-one,sulfonyl-1,4-dihydro-1,2,4-triazol-5-one, sulfanyl 1H-tetrazole,sulfanyl 2H-tetrazole, sulfinyl 1H-tetrazole, sulfinyl 2H-tetrazole,sulfonyl 1H-tetrazole, sulfonyl 2H-tetrazole, and sulfonimidamide.

In certain embodiments of a compound of Formula (1), the acidicoxocarbocycle or cyclic polyone and resonance forms is selected fromcyclopentane-1,3-dione, squaric acid, squareamide, mixed squaramate, and2,6-difluorophenol.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —SO₂OH, —P(O)(OH)R¹², —P(O)(OH)(OR¹²), —SO₂NHR¹²,—NHSO₂R¹², —SO₂NHCOR¹², —CONHSO₂R¹², —SO₂NHCONHR¹², —CONHCN,1H-tetrazol-yl, 5-oxo-1,2,4-oxadiazole, 5-oxo-1,2,4-thiadiazole,5-thioxo-1,2,4-oxadiazole, thiazolidinedione, oxazolidinedione,oxadiazolidinedione, 3-hydroxyisoxazole, 3-hydroxyisothiazole,cyclopentane-1,3-dione, squaric acid, squareamide, and mixed squaramate;wherein R¹² is selected from hydrogen, C₁₋₄ alkyl, and C₃₋₅ cycloalkyl.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —P(O)(OH)H, —CONHSO₂CH₃, —CONHSO₂CF₃, —SO₂NHCOCH₃,—SO₂NHCOCF₃, —NHSO₂CH₃, —NHSO₂CF₃, 1H-tetrazol-yl,5-oxo-1,2,4-oxadiazole-yl, 5-oxo-1,2,4-thiadiazole-yl,5-thioxo-1,2,4-oxadiazole-yl, thiazolidinedione-yl, oxazolidinedione-yl,oxadiazolidinedione-yl, 3-hydroxyisoxazole-yl, 3-hydroxyisothiazole-yl,tetronic acid-yl, tetramic acid-yl, and cyclopentane-1,3-dione-yl.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —P(O)(OH)H, —CONHSO₂CH₃, —CONHSO₂CF₃, —SO₂NHCOCH₃,—SO₂NHCOCH₃, —SO₂NHCOCF₃, —NHSO₂CF₃, —NHSO₂CF₃, and 1H-tetrazol-5-yl.

In certain embodiments of a compound of Formula (1), R⁶ is selected from—COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazol-yl.

In certain embodiments of a compound of Formula (1), R⁶ is —COOH.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen, deuterio, halogen, hydroxyl, andC₁₋₄ alkyl, or two germinal R⁷ together with the carbon atom to whichthey are bonded form a C₃₋₅ cycloalkyl ring.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen, deuterio, fluoro, hydroxyl,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl, ortwo germinal R⁷ together with the carbon atom to which they are bondedform a cyclopropyl ring or a cyclobutyl ring.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen, deuterio, fluoro, hydroxyl, andmethyl.

In certain embodiments of a compound of Formula (1), each R⁷ isindependently selected from hydrogen and deuterio.

In certain embodiments of a compound of Formula (1), each R⁷ ishydrogen.

In certain embodiments of a compound of Formula (1), R⁸ is selected fromhydrogen, deuterio, halogen, hydroxyl, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, and cyclopropyl.

In certain embodiments of a compound of Formula (1), R⁸ is selected fromhydrogen, deuterio, halogen, hydroxyl, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy,isopropoxy, trifluoromethoxy, and cyclopropyl.

In certain embodiments of a compound of Formula (1), R⁸ is selected fromhydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, tert-butyl,hydroxyl, methoxy, ethoxy, isopropoxy, trifluoromethyl, andtrifluoromethoxy.

In certain embodiments of a compound of Formula (1), R⁸ is methyl.

In certain embodiments of a compound of Formula (1), R⁸ is hydrogen.

In certain embodiments of a compound of Formula (1), each R¹⁰ isindependently selected from hydrogen and C₁₋₄ alkyl, or two R¹⁰ togetherwith the nitrogen atom to which they are bonded form a 3- to 5-memberedheterocycle.

In certain embodiments of a compound of Formula (1), L is (—X—)_(a)wherein a is selected from 0, 1, 2, 3, and 4, and X is selected fromoxygen (—O—), sulfur (—S—), sulfinyl (—SO—), sulfonyl (—SO₂—), carbonyl(—CO—), —C(R¹⁶)₂— wherein R¹⁶ is independently selected from hydrogen,deuterio, halogen, hydroxyl, and C₁₋₄ alkyl, and amino (—NR¹⁷—), whereinR¹⁷ is selected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1), comprises isselected from a bond (“—”), methylene (—CH₂—), fluoromethylene (—CFH—),difluoromethylene (—CF₂—), hydroxymethylene (—C(OH)H—), ethane-1,1-diyl(—CHCH₃—), propane-2,2-diyl (—C(CH₃)₂—), propane-1,1-diyl(—CH(CH₂—CH₃)—), oxygen (—O—), sulfur (—S—), sulfinyl (—SO—), sulfonyl(—SO₂—), carbonyl (—CO—), and amino (—NR¹⁷—), wherein R¹⁷ is selectedfrom hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1), comprises isselected from a bond (“—”), methylene (—CH₂—), fluoromethylene (—CFH—),difluoromethylene (—CF₂—), hydroxymethylene (—C(OH)H—), ethane-1,1-diyl(—CHCH₃—), propane-2,2-diyl (—C(CH₃)₂—), oxygen (—O—), sulfonyl (—SO₂—),carbonyl (—CO—), and amino (—NR¹⁷—), wherein R¹⁷ is selected fromhydrogen and methyl.

In certain embodiments of a compound of Formula (1), a is 2 and each Xis methylene (—CH₂—) and L is ethane-1,2-diyl (—CH₂—CH₂—); one X ismethylene (—CH₂—) and one X is ethane-1,1-diyl (—CHCH₃—) and L ispropane-1,2-diyl (—CH₂—CHCH₃—); one X is ethane-1,1-diyl (—CHCH₃—) andone X is methylene (—CH₂—) and L is propane-1,2-diyl (—CHCH₃—CH₂—); oneX is methylene (—CH₂—) and one X is hydroxymethylene (—CHOH—) and L ishydroxyethane-1,2-diyl (—CH₂—CHOH—); one X is hydroxymethylene (—CHOH—)and one X is methylene (—CH₂—) and L is hydroxyethane-1,2-diyl(—CHOH—CH₂—); one X is methylene (—CH₂—) and one X is fluoromethylene(—CFH—), and L is fluoroethane-1,2-diyl (—CH₂—CHF—); one X isfluoromethylene (—CFH—) and one X is methylene (—CH₂—) and L isfluoroethane-1,2-diyl (—CHF—CH₂—); one X is methylene (—CH₂—) and one Xis difluoromethylene (—CF₂—), and L is difluoroethane-1,2-diyl(—CH₂—CF₂—); one X is difluoromethylene (—CF₂—) and one X is methylene(—CH₂—) and L is difluoroethane-1,2-diyl (—CF₂—CH₂—); one X is carbonyl(—CO—) and one X is amino (—NR¹⁷—) and L is carbonyl amino (—CO—NR¹⁷—);one X is amino (—NR¹⁷—) and one X is carbonyl (—CO—) and L is aminocarbonyl (—NR¹⁷—CO—); one X is methylene (—CH₂—) and one X is amino(—NR¹⁷—) and L is methyleneamino (—CH₂—NR¹⁷—); one X is amino (—NR¹⁷—)and one X is methylene (—CH₂—) and L is aminomethylene (—NR¹⁷—CH₂—); oneX is methylene (—CH₂—) and one X is oxygen (—O—) and L is methyleneoxy(—CH₂—O—); one X is oxygen (—O—) and one X is methylene (—CH₂—) and L isoxymethylene (—O—CH₂—); one X is methylene (—CH₂—) and one X is sulfur(—S—) and L is methylenethiyl (—CH₂—S—); one X is sulfur (—S—) and one Xis methylene (—CH₂—) and L is thiylmethylene (—S—CH₂—); one X ismethylene (—CH₂—) and one X is sulfinyl (—SO—) and L ismethylenesulfinyl (—CH₂—SO—); one X is sulfinyl (—SO—) and one X ismethylene (—CH₂—) and L is sulfinylmethylene (—SO—CH₂—); one X ismethylene (—CH₂—) and one X is sulfonyl (—SO₂—) and L ismethylenesulfonyl (—CH₂—SO₂—); one X is sulfonyl (—SO₂—) and one X ismethylene (—CH₂—) and L is sulfonylmethylene (—SO₂—CH₂—); one X ismethylene (—CH₂—) and one X is carbonyl (—CO—) and L ismethylenecarbonyl (—CH₂—CO—); or one X is carbonyl (—CO—) and one X ismethylene (—CH₂—) and L is carbonylmethylene (—CO—CH₂—); wherein R¹⁷ isselected from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1), a is 2 and L isselected from ethane-1,2-diyl (—CH₂—CH₂—), propane-1,2-diyl (—CH₂—CHCH₃—or —CHCH₃—CH₂—), hydroxyethane-1,2-diyl (—CH₂—CHOH— or —CHOH—CH₂—),carbonyl amino (—CO—NR¹⁷—), amino carbonyl (—NR¹⁷—CO—), methyleneamino(—CH₂—NR¹⁷—), aminomethylene (—NR¹⁷—CH₂—), methyleneoxy (—CH₂—O—),oxymethylen (—O—CH₂—), methylenethiyl (—CH₂—S—), thiylmethylene(—S—CH₂—), methylenesulfonyl (—CH₂—SO₂—), sulfonylmethylene (—SO₂—CH₂—),methylenecarbonyl (—CH₂—CO—), and carbonylmethylene (—CO—CH₂—), whereinR¹⁷ is selected from hydrogen and methyl.

In certain embodiments of a compound of Formula (1),

at least one of R¹ and R⁵ is independently selected from, halogen,—N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰,—CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, substituted C₁₋₄ alkyl, C₁₋₄ alkoxy,substituted C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyland C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1), at least one of R¹and R⁵ is independently selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰),—NO₂, —NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, andC₃₋₅ cycloalkyloxy;

each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyland C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1), each of R¹ and R⁵is independently selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂,—NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, andC₃₋₅ cycloalkyloxy;

each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring; and

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl;

each R¹⁰ is independently selected from hydrogen, deuterio, C₁₋₄ alkyland C₁₋₄ alkoxy, or two geminal R¹⁰ together with the nitrogen to whichthey are bonded form a 3- to 6-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy,C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;

each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring; and

one of R¹, R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

In certain embodiments of a compound of Formula (1),

each of the other of R¹, R², R³, R⁴, and R⁵ is independently is selectedfrom hydrogen, deuterio, halogen, —N(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO,—OH, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl,C₁₋₄ alkylsulfonyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₄ alkyl,C₁₋₄ alkoxy, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, and C₄₋₈cycloalkylalkyl; and

each R¹⁰ is independently selected from hydrogen and C₁₋₄ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to6-membered heterocyclic ring.

In certain embodiments of a compound of Formula (1),

each of the other of R¹, R², R³, R⁴, and R⁵ is independently selectedfrom hydrogen, deuterio, halogen, —NR¹⁰ ₂, —N(R¹⁰)(OR¹⁰), —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ fluoroalkyl, and C₁₋₄fluoroalkoxy, and

each R¹⁰ is independently selected from hydrogen and C₁₋₄ alkyl, or twoR¹⁰ together with the nitrogen to which they are bonded form a 3- to5-membered heterocyclic ring.

In certain embodiments of a compound of Formula (1), the other of R¹ andR⁵ is hydrogen.

In certain embodiments of a compound of Formula (1), each of the otherof R¹, R², R³, R⁴, and R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1), R², R³, and R⁵ ishydrogen.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(OR¹⁰)(R¹⁰),—NO₂, —NO, —N(R¹⁰)(S(═O)R¹⁰), —N(R¹⁰)(S(═O)₂R¹⁰), —N(R¹⁰)(C(O)R¹⁰),—N(R¹⁰)(C(O)OR⁰), —N(R¹⁰)(C(O)N(R¹⁰)₂, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,—SH, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl,—S(O)N(R¹⁰)₂, —S(O)₂N(R¹⁰)₂, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₁₋₆alkyl, substituted C₁₋₆ alkyl, C₁₋₆ alkoxy, substituted C₁₋₆ alkoxy,C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyloxy,substituted C₃₋₆ cycloalkyloxy, C₄₋₁₂ cycloalkylalkyl, substituted C₄₋₁₂cycloalkylalkyl, C₆₋₁₀ aryl, substituted C₆₋₁₀ aryl, C₇₋₁₆ arylalkyl,substituted C₇₋₁₆ arylalkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₁₋₆ heteroalkoxy, substituted C₁₋₆ heteroalkoxy, C₃₋₆heterocycloalkyl, substituted C₃₋₆ heterocycloalkyl, C₄₋₁₂heterocycloalkylalkyl, substituted C₄₋₁₂ heterocycloalkylalkyl, C₅₋₁₀heteroaryl, substituted C₅₋₁₀ heteroaryl, C₆₋₁₆ heteroarylalkyl, andsubstituted C₆₋₁₆ heteroarylalkyl; wherein each R¹⁰ is independentlyselected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄ alkoxy, or twogeminal R¹⁰ together with the nitrogen to which they are bonded form a3- to 6-membered heterocyclic ring; and

R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1),

R¹ selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂,—NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl, and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring; and

R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl,or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring; and

R⁵ is hydrogen.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰), —NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH,C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄alkylsulfonyl, C₁₋₄ heteroalkyl, C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl,C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, C₃₋₅ cycloalkyloxy, and C₄₋₈cycloalkylalkyl; wherein each R¹⁰ is independently selected fromhydrogen, deuterio, C₁₋₄ alkyl and C₁₋₄ alkoxy, or two geminal R¹⁰together with the nitrogen to which they are bonded form a 3- to6-membered heterocyclic ring;

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R², R³, and R⁴ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—SO₂—CH₂—, —CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected fromhydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

each of R¹ and R⁵ is independently selected from halogen, —N(R¹⁰)₂,—NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy,C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy; wherein each R¹⁰ isindependently selected from hydrogen and C₁₋₃ alkyl, or two R¹⁰ togetherwith the nitrogen to which they are bonded form a 3- to 5-memberedheterocyclic ring;

one of R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of the other R², R³, and R⁴ is hydrogen;

R⁶ is —COOH;

each R⁷ is selected from hydrogen, methyl, hydroxyl, and fluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰),—NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

one of R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—SO₂—CH₂—, —CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected fromhydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl,or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring;

one of R², R³, R⁴, and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of the other of R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is —COOH;

each R⁷ is selected from hydrogen, methyl, hydroxyl, and fluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

R⁵ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰),—NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

one of R¹, R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, and R⁴ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy;

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—SO₂—CH₂—, —CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected fromhydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

R⁵ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen and C₁₋₃ alkyl,or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring;

one of R¹, R², R³, and R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, and R⁴ is hydrogen;

R⁶ is —COOH;

each R⁷ is selected from hydrogen, methyl, hydroxyl, and fluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy;

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O—)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—SO₂—CH₂—, —CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected fromhydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

one of R¹ and R⁵ is selected from —N(—CH₂—CH₂—R⁹)₂,—CH₂—N(—CH₂—CH₂—R⁹)₂, —N⁺(—O—)(—CH₂—CH₂—R⁹)₂,—CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —O—N(—CH₂—CH₂—R⁹)₂,—CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂, —CH₂—CO—N(—CH₂—CH₂—R⁹)₂,—O—CO—N(—CH₂—CH₂—R⁹)₂, and —CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ isindependently selected from —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃;

each of the other of R¹, R², R³, R⁴, and R⁵ is hydrogen;

R⁶ is —COOH;

each R⁷ is selected from hydrogen, methyl, hydroxyl, and fluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —N⁺(—O⁻)(R¹⁰)₂, —N(R¹⁰)(OR¹⁰),—NO₂, —NO, —CN, —COOR¹⁰, —CON(R¹⁰)₂, —OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄ alkylsulfonyl, C₁₋₄ heteroalkyl,C₁₋₄ heteroalkoxy, C₁₋₄ fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl,C₃₋₅ cycloalkyloxy, and C₄₋₈ cycloalkylalkyl; wherein each R¹⁰ isindependently selected from hydrogen, deuterio, C₁₋₄ alkyl and C₁₋₄alkoxy, or two geminal R¹⁰ together with the nitrogen to which they arebonded form a 3- to 6-membered heterocyclic ring;

R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂, —CH₂—N(—CH₂—CH₂—R⁹)₂,—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —CH₂—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂,—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—O—N(—CH₂—CH₂—R⁹)₂, —CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂,—CH₂—CO—N(—CH₂—CH₂—R⁹)₂, —O—CO—N(—CH₂—CH₂—R⁹)₂, and—CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ is independently selectedfrom —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of R², R³, and R⁵ is hydrogen;

R⁶ is selected from —COOH, —S(O)OH, —P(O)(OH)H, and 1H-tetrazole;

each R⁷ is independently selected from hydrogen, methyl, hydroxyl, andfluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, cyclobutyl, tert-butyl, hydroxyl, C₁₋₄alkoxy, C₁₋₄ fluoroalkyl, and C₁₋₄ fluoroalkoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —SO₂—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—SO₂—CH₂—, —CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected fromhydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (1),

R¹ is selected from halogen, —N(R¹⁰)₂, —NR¹⁰(OR¹⁰), —NO₂, —NO, —OH, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylsulfanyl, C₁₋₄ alkylsulfinyl, C₁₋₄fluoroalkyl, C₁₋₄ fluoroalkoxy, C₃₋₅ cycloalkyl, and C₃₋₅ cycloalkyloxy;wherein each R¹⁰ is independently selected from hydrogen or C₁₋₃ alkyl;or two R¹⁰ together with the nitrogen to which they are bonded form a 3-to 5-membered heterocyclic ring;

R⁴ is selected from —N(—CH₂—CH₂—R⁹)₂, —CH₂—N(—CH₂—CH₂—R⁹)₂,—N⁺(—O⁻)(—CH₂—CH₂—R⁹)₂, —CH₂—N(—O—)(—CH₂—CH₂—R⁹)₂,—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹), —CH₂—N(—O—CH₂—CH₂—R⁹)(—CH₂—CH₂—R⁹),—O—N(—CH₂—CH₂—R⁹)₂, —CH₂—O—N(—CH₂—CH₂—R⁹)₂, —CO—N(—CH₂—CH₂—R⁹)₂,—CH₂—CO—N(—CH₂—CH₂—R⁹)₂, —O—CO—N(—CH₂—CH₂—R⁹)₂, and—CH₂—O—CO—N(—CH₂—CH₂—R⁹)₂, wherein each R⁹ is independently selectedfrom —Cl, —Br, —I, —OSO₂CH₃, and —OSO₂CF₃.

each of R², R³, and R⁵ is hydrogen;

R⁶ is —COOH;

each R⁷ is selected from hydrogen, methyl, hydroxyl, and fluoro;

R⁸ is selected from hydrogen, methyl, ethyl, propyl, isopropyl,cyclopropyl, tert-butyl, hydroxyl, methoxy, ethoxy, isopropoxy,trifluoromethyl, and trifluoromethoxy; and

L is selected from a bond “—”, —CH₂—, —C(OH)H—, —CHCH₃—, —C(CH₃)₂—,—CF₂—, —O—, —NR¹⁷—, —CO—, —CH₂—CH₂—, —CH₂—CHCH₃—, —CHCH₃—CH₂—,—CH₂—CHOH—, —CHOH—CH₂—, —CH₂—CF₂—, —CF₂—CH₂—, —CO—NR¹⁷—, —NR¹⁷—CO—,—CH₂—NR¹⁷—, —NR¹⁷—CH₂—, —CH₂—O—, —O—CH₂—, —CH₂—S—, —S—CH₂—, —CH₂—SO₂—,—CH₂—CO—, and —CO—CH₂—, wherein R¹⁷ is selected from hydrogen andmethyl.

In certain embodiments, R⁸ is selected from hydrogen, deuterio, C₁₋₆alkyl, substituted C₁₋₆ alkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl, C₃₋₆ cycloalkyl, substituted C₃₋₆ cycloalkyl, —COOR¹⁰, C₁₋₄fluoroalkyl, C₃₋₆ cycloalkyl, and phenyl;

In certain embodiments, R⁸ is selected from hydrogen, deuterio, C₁₋₄alkyl, C₁₋₄ fluoroalkyl, and cyclopropyl.

In certain embodiments, R⁸ is selected from hydrogen, deuterio, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, trifluoromethyl,and cyclopropyl.

In certain embodiments, L is —(X)_(a)—, wherein, each X is independentlyselected from a bond (“—”), —C(R¹⁶)₂—, wherein each R¹⁶ is independentlyselected from hydrogen, deuterio, halogen, hydroxyl, C₁₋₄ alkyl and C₁₋₄alkoxy, or two R¹⁶ together with the carbon to which they are bondedform a C₃₋₆ cycloalkyl ring or a C₃₋₆ heterocycloalkyl ring, —O—, —S—,—SO—, —SO₂—, —CO—, and —N(R¹⁷)—, wherein R¹⁷ is selected from hydrogenand C₁₋₄ alkyl; and a is selected from 0, 1, 2, 3, and 4;

In certain embodiments, L is selected from a bond (“—”), methylene(—CH₂—), fluoromethylene (—CFH—), difluoromethylene (—CF₂—),hydroxymethylene (—C(OH)H—), ethane-1,1-diyl (—CHCH₃—), propane-2,2-diyl(—C(CH₃)₂—), propane-1,1-diyl (—CH(CH₂—CH₃)—), sulfinyl (—SO—), sulfonyl(—SO₂—), and carbonyl (—CO—).

In certain embodiments, L is selected from a bond (“—”), methylene(—CH₂—), fluoromethylene (—CFH—), difluoromethylene (—CF₂—),hydroxymethylene (—C(OH)H—), ethane-1,1-diyl (—CHCH₃—), propane-2,2-diyl(—C(CH₃)₂—), sulfonyl (—SO₂—), and carbonyl (—CO—).

In certain embodiments, L is selected from ethane-1,2-diyl (—CH₂—CH₂—),propane-1,2-diyl (—CH₂—CHCH₃ or —CHCH₃—CH₂—), hydroxyethane-1,2-diyl(—CH₂—CHOH— or —CHOH—CH₂—), fluoroethane-1,2-diyl (—CH₂—CHF— or—CHF—CH₂—), difluoroethane-1,2-diyl (—CH₂—CF₂— or —CF₂—CH₂—), carbonylamino (—CO—NR¹⁷—), methyleneamino (—CH₂—NR¹⁷—), methyleneoxy (—CH₂—O—),methylenethiyl (—CH₂—S—), methylenesulfinyl (—CH₂—SO—),sulfinylmethylene (—SO—CH₂—), methylenesulfonyl (—CH₂—SO₂—),sulfonylmethylene (—SO₂—CH₂—), methylenescarbonyl (—CH₂—CO—), andcarbonylmethylene (—CO—CH₂—), wherein R¹⁷ is selected from hydrogen,methyl, and ethyl.

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the beta-carbon atom is (R).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the beta-carbon atom is (S).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the 3 carbon atom is of the (R) configuration, theabsolute axial stereochemistry (atropisomerism) is R_(a), and theabsolute stereochemistry of a compound of Formula (1) is (R,R_(a)).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the β-carbon atom is of the (R) configuration, theabsolute axial stereochemistry (atropisomerism) is S_(a), and theabsolute stereochemistry of a compound of Formula (1) is (R,S_(a)).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the β-carbon atom is of the (S) configuration, theabsolute axial stereochemistry (atropisomerism) is R_(a), and theabsolute stereochemistry of a compound of Formula (1) is (S,R_(a)).

In certain embodiments of a compound of Formula (1), the absolutestereochemistry of the β-carbon atom is of the (S) configuration, theabsolute axial stereochemistry (atropisomerism) is S_(a), and theabsolute stereochemistry of a compound of Formula (1) is (S,S_(a)).

In certain embodiments, a compound of Formula (1) is selected from:

-   3-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoic    acid (1);-   3-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoic    acid (2);-   3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid    (3);-   3-Amino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid    (4);-   (3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic    acid (5);-   (3R)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic    acid (6);-   (3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoic    acid (7);-   (3S)-3-Amino-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoic    acid (8);-   (3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoic    acid (9);-   [(2R)-2-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinic    acid (10);-   (3R)-3-Amino-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic    acid (11);-   (3R)-3-Amino-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic    acid (12);-   (3R)-3-Amino-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic    acid (13);-   (3R)-3-Amino-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoic    acid (14);-   (3R)-3-Amino-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoic    acid (15);-   (3S)-3-Amino-4-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]amino]-4-oxo-butanoic    acid (16);-   (3S)-3-Amino-4-[2-[bis(2-chloroethyl)amino]phenoxy]butanoic acid    (17);-   (3R)-3-Amino-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]pentanoic    acid (18);-   (3R)-3-Amino-4-[5-(2-chloroethyl(chloromethyl)carbamoyl)oxy-2-methyl-phenyl]butanoic    acid (19);-   (3R)-3-Amino-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]butanoic    acid (20);-   (3R)-3-amino-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoic    acid (21);-   3-[(2R)-2-Amino-4-hydroxy-4-oxo-butyl]-N,N-bis(2-chloroethyl)-4-methyl-benzeneamine    oxide (22); and-   (3R)-3-Amino-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]butanoic acid    (23);-   or a pharmaceutically acceptable salt or salts of any of the    foregoing.

In certain embodiments of any of the foregoing compounds, apharmaceutically acceptable salt is the hydrochloride salt.

In certain embodiments of any of the foregoing compounds, apharmaceutically acceptable salt is the dihydrochloride salt.

In certain embodiments of a compound of Formula (1), a pharmaceuticallyacceptable salt is the hydrochloride salt.

In certain embodiments of a compound of Formula (1), a pharmaceuticallyacceptable salt is the dihydrochloride salt.

In certain embodiments, compounds of Formula (1) are selectivesubstrates for the LAT1/4F2hc transporter.

In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent V_(max) of at least 10% the V_(max) ofgabapentin. In certain embodiments, compounds provided by the presentdisclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least 20% theV_(max) of gabapentin. In certain embodiments, compounds provided by thepresent disclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least30% the V_(max) of gabapentin. In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependentV_(max) of at least 40% the V_(max) of gabapentin. In certainembodiments, compounds provided by the present disclosure exhibit aLAT1/4F2hc-dependent V_(max) of at least 50% the V_(max) of gabapentin.In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent V_(max) of at least 60% the V_(max) ofgabapentin. In certain embodiments, compounds provided by the presentdisclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least 70% theV_(max) of gabapentin. In certain embodiments, compounds provided by thepresent disclosure exhibit a LAT1/4F2hc-dependent V_(max) of at least80% the V_(max) of gabapentin. In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependentV_(max) of at least 90% the V_(max) of gabapentin. In certainembodiments, compounds provided by the present disclosure exhibit aLAT1/4F2hc-dependent V_(max) of at least 100% the V_(max) of gabapentin.

In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L) and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 50% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L). In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependent uptakeof at least 10% that of gabapentin measured at an extracellularconcentration of 1 mM (1 mmol/L); and a system A-, system N-, a systemASC-, and a LAT2/4F2hc-dependent uptake of less than 40% that ofL-leucine measured at an extracellular concentration of 1 mM (1 mmol/L).In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 30% that of L-leucine measured at an extracellularconcentration of 1 mM (mmol/L). In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependent uptakeof at least 10% that of gabapentin measured at an extracellularconcentration of 1 mM (1 mmol/L); and a system A-, system N-, a systemASC-, and a LAT2/4F2hc-dependent uptake of less than 20% that ofL-leucine measured at an extracellular concentration of 1 mM (1 mmol/L).In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 10% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L). In certain embodiments, compoundsprovided by the present disclosure exhibit a LAT1/4F2hc-dependent uptakeof at least 10% that of gabapentin measured at an extracellularconcentration of 1 mM (1 mmol/L); and a system A-, system N-, a systemASC-, and a LAT2/4F2hc-dependent uptake of less than 5% that ofL-leucine measured at an extracellular concentration of 1 mM (1 mmol/L).In certain embodiments, compounds provided by the present disclosureexhibit a LAT1/4F2hc-dependent uptake of at least 10% that of gabapentinmeasured at an extracellular concentration of 1 mM (1 mmol/L); and asystem A-, system N-, a system ASC-, and a LAT2/4F2hc-dependent uptakeof less than 1% that of L-leucine measured at an extracellularconcentration of 1 mM (1 mmol/L).

Compounds of Formula (1) may be adapted as prodrugs to achieve desirablepharmacokinetic properties. For example, suitable prodrugs ofβ-substituted β-amino acid derivatives and β-substituted β-amino acidanalogs are disclosed by Gallop, et al., U.S. Pat. No. 7,109,239, U.S.Pat. No. 6,972,341, U.S. Pat. No. 6,818,787 and U.S. Pat. No. 7,227,028.Prodrugs of compounds of Formula (1) include the prodrug systemsdisclosed by Gallop, et al., as well as others known in the art.

Synthesis of Compounds

Compounds disclosed herein may be obtained via the general syntheticmethods illustrated in Schemes 1-10. General synthetic methods useful inthe synthesis of compounds, precursors, and starting materials describedherein are available in the art. Starting materials useful for preparingcompounds and intermediates thereof, and/or practicing methods describedherein, are commercially available or may be prepared by well-knownsynthetic methods (March's Advanced Organic Chemistry: Reactions,Mechanisms, M. B. Smith, 7^(th) Edition, John Wiley & Sons, Hoboken,N.J., USA, 2013; Advanced Organic Chemistry: Part B: Reaction andSynthesis, F. A. Carey and R. J. Sundberg, 5^(th) Edition, Springer,Germany, 2010; Comprehensive Organic Transformations, 2^(nd) Edition,and R. C. Larock, Wiley-VCH, Weinheim, Germany, 1999).

Additionally, as will be apparent to those skilled in the art, use ofconventional protecting groups or protecting strategies may be necessaryto prevent certain functional groups from undergoing undesiredreactions. Suitable protecting groups for various functional groups aswell as suitable conditions for protecting and deprotecting particularfunctional groups are well known in the art. On the other hand, manymethods for selective removal of protecting groups without affecting thedesired molecular architecture are also well known in the art (Wuts andGreene, Greene's Protective Groups in Organic Synthesis, 4^(th) Ed,2007, Wiley-Interscience, John Wiley & Sons, Inc., Hoboken, N.J.).

It will be appreciated that where typical or preferred processconditions, e.g., reaction temperatures, reaction times, molar ratios ofreactants, solvents, pressures, etc., are given other process conditionsmay also be used. Optimal reaction conditions may vary with theparticular reactants, solvents, functional groups, and protecting groupsused, but such conditions may be determined by one skilled in the art byroutine optimization procedures.

Furthermore, certain compounds provided by the present disclosure maycontain one or more stereogenic centers. Accordingly, and if desired,such compounds may be prepared or isolated as pure stereoisomers, e.g.,as individual enantiomers, diastereomers, atropisomers, rotamers, or asstereoisomer enriched mixtures or racemates. All such stereoisomers areincluded within the scope of this disclosure. Pure stereoisomers (orenriched mixtures thereof) may be prepared using, for example, opticallyactive starting materials, stereoselective reagents such as chiralcatalysts and auxiliaries well known in the art. Alternatively, racemicmixtures of such compounds may be separated or partially enriched using,for example, chromatographic methods with chiral stationary phases,chiral resolving agents, and the like, also well known in the art andeasily adaptable to the particular compound to be separated.

There has been an ever growing interest in the synthesis of β-aminoacids with various substitution patterns. Depending on the location andthe number of the substitutents, β-amino acids are categorized as (a)β²-(mono-α-substituted), (b) β³-(mono-β-substituted), (c)β^(2,3)-(α,β-di-substituted), (d) β^(2,2)-(α,α-di-substituted orα-geminal-disubstituted), (e) β^(3,3)-(β,β-di-substituted orβ-geminal-disubstituted), (f) β^(2,2,3)-(α,α,β-tri-substituted), (g)β^(2,3,3)-(α,β,β-tri-substituted), or (h)β^(2,2,3,3)-((α,α,β,β-tetra-substituted) amino acids. Many methods forthe synthesis of protected and unprotected β-amino acids with a widevariety of type and number of substituents either in racemic, enatio- ordiastereomerically enriched or pure form from commercial or knownstarting materials are well known in the art (Enantioselective Synthesisof β-Amino Acids, 2^(nd) Edition, E. Juaristi and V. Soloshonok, JohnWiley & Sons, 2005, Hoboken, N.J., USA, 2005; Smith, Methods ofNon-α-Amino Acid Synthesis, Marcel Dekker, Inc., New York, USA, 1995;Cole, Tetrahedron, 1994, 50 (32), 9517-9582; Juaristi, et al., AldrichChim. Acta, 1994, 27(1), 3-11; Lelais and Seebach, Biopolymers (PeptideScience), 2004, 76, 206-243; Sewald, Amino Acids, 1996, 11, 397-408;Seebach, et al., Synthesis, 2009, (1), 1-32; and Abele and Seebach, Eur.J. Org. Chem., 2000, (1), 1-15).

In particular, many methods of preparing protected and unprotectedβ³-substituted racemic or optically active β-amino acids, β-amino acidsanalogs, or β-amino acid carboxylic acid (bio)isosters from commercialor known starting materials are well known in the art.

In certain embodiments, such derivatives may be used as convenientstarting materials for the preparation of the target compounds providedby the present disclosure. In certain embodiments, suitablyfunctionalized protected and unprotected β³-substituted racemic oroptically active β-amino acids, β-amino acids analogs, or β-amino acidcarboxylic acid (bio)isosters may be used as starting materials for thepreparation of the target compounds provided by the present disclosure.

In certain embodiments, starting materials may be used in their fullyprotected form wherein the amino group or a synthetic equivalent or aprecursor thereof and the carboxylic acid, phosphinic acid, sulfinicacid, carboxylic acid (bio)isosteres or synthetic equivalents orprecursors of any of the foregoing are appropriately protected.

In certain embodiments, starting materials may be used in theirhemi-protected form wherein the amino group or a synthetic equivalent ora precursor thereof is protected and the carboxylic acid group,phosphinic acid, sulfinic acid, or carboxylic acid (bio)isosterefunctional group or synthetic equivalents or precursors of any of theforegoing are unprotected or free.

In certain embodiments, starting materials may be used in theirhemi-protected form wherein the amino group is unprotected or free andthe carboxylic acid, phosphinic acid, sulfinic acid, or carboxylic acid(bio)isostere or synthetic equivalents or precursors of any of theforegoing are appropriately protected.

In certain embodiments, starting materials may be used in their fullunprotected form wherein the amino group and the carboxylic acid, freephosphinic acid, free sulfinic acid, or free carboxylic acid(bio)isostere or synthetic equivalents or precursors of any of theforegoing are unprotected.

In certain embodiments, protected and unprotected β³-substituted racemicor optically active β-amino acids, β-amino acids analogs, or β-aminoacid carboxylic acid (bio)isosters bear a chemical functional grouplinking the β³-carbon atom to an aromatic ring system. In certainembodiments, the aromatic ring system is functionalized with ananchoring group in order to install a chemotherapeutic moiety.

Methods of synthetic manipulations and modifications of the underlyingprotected or unprotected β-amino acid scaffold are well known in theart. In certain embodiments, the underlying the underlying β-amino acidscaffold may be modified to allow for regio- and/or stereoselectiveincorporation of auxiliary molecular functionalities. Auxiliarymolecular functionalities may, for example, be incorporated to modulateinteraction with LAT1 transporter proteins, e.g., efficacy oftranslocation through biological membranes (binding to theLAT1-transporter protein and capacity of LAT1-mediated transport), aidthe modulation of physiochemical parameter, or to modulate the activityof the physiologically active N-mustard moiety, e.g., cytotoxicity.

In certain embodiments, the underlying aryl-ring may be modified toallow for regioselective incorporation of functional groups that can beconverted to chemotherapeutic moieties by using reagents, methods, andprotocols well known in the art.

In certain embodiments, the underlying aryl-ring may be modified toallow for regio- and/or stereoselective incorporation of auxiliarymolecular functionalities into the arene scaffold. Auxiliary molecularfunctionalities may, for example, be incorporated to modulateinteraction with LAT1 transporter proteins, e.g., efficacy oftranslocation through biological membranes (binding to theLAT1-transporter protein and capacity of LAT1-mediated transport), or tomodulate the activity of the physiologically active chemotherapeuticmoiety, e.g., cytotoxicity.

Many other methods for the preparation of appropriately functionalizedor substituted, protected and unprotected β³-substituted racemic oroptically active β-amino acids, β-amino acids analogs, or β-amino acidcarboxylic acid (bio)isosters, derivatives or precursors of any of theforegoing from commercial or known starting materials and employingmethods and protocols are either described herein, are described in theart, or will be readily apparent to the one skilled in the art.Accordingly, the methods presented in the schemes provided by thepresent disclosure are illustrative rather than comprehensive.

Referring to Scheme 1, selected and representative starting materialsfor the preparation N-mustard functionalized β-branched β-amino acids,β-amino acid analogs, or β-amino acids carboxylic acid (bio)isosteresare compounds of Formula (A). This selection is not intended to belimiting in any way.

Referring to Scheme 1, in certain embodiments R¹ and/or R⁵, and thelinker L are defined as described herein; one of R², R³, and R⁴ incompounds of Formula (A) is -E-MH, wherein E is a bond (“—”), an oxygenatom (—O—), a methylene group (—CH₂—), a methyleneoxy group (—CH₂—O—), acarbonyl group (—CO—), or a methylenecarbonyl group (—CH₂—CO—), andwherein MH is an amino group (—NH₂), a hydroxyl group (—OH), or asulfhydryl group (—SH). Each of the other remaining R², R³, and R⁴ ishydrogen; each R⁷ and each R⁸ is hydrogen.

Referring to Scheme 1, for example, (a) -E-MH is equivalent to a primaryaromatic amino group (—NH₂, aniline) when E is a bond (“—”) and MH is anamino group (—NH₂), (b) -E-MH is equivalent to a primary O-arylhydroxylamino group (—O—NH₂) when E is an oxygen atom (—O—) and MH is anamino group (—NH₂), (c) -E-MH is equivalent to a primary aminomethylgroup (—CH₂—NH₂, primary benzylic amine) when E is a methylene group(—CH₂—) and MH is an amino group (—NH₂), (d) -E-MH is equivalent to anaromatic hydroxyl group (—OH, phenol) when E is a bond (“—”) and MH is ahydroxyl group (—OH), (e) -E-MH is equivalent to a hydroxymethyl group(—CH₂—OH, benzylic alcohol) when E is a methylene group (—CH₂—) and MHis a hydroxyl group (—OH), (f) -E-MH is equivalent to a primaryO-benzylic hydroxylamino group (—CH₂—O—NH₂) when E is a methyleneoxygroup (—CH₂—O—) and MH is an amino group (—NH₂), (g) -E-MH is equivalentto an aromatic sulhydryl group (—SH, thiophenol derivative) when E is abond (“—”) and MH is a hydroxyl group (—OH), (h) -E-MH is equivalent toa methylenesulhydryl group (—CH₂—SH, benzylic thiol) when E is amethylene group (—CH₂—) and MH is a sulfhydryl group (—SH), (i) -E-MH isequivalent to an aromatic carboxylic acid group (—CO—OH, benzoic acid)when E is a carbonyl group (—C(═O)—) and MH is a hydroxyl group (—OH),(j) -E-MH is equivalent to a carboxylic acid group (—CO—OH, benzoicacid) when E is a methylenecarbonyl group (—CH₂—C(═O)—) and MH is ahydroxyl group (—OH).

It will be understood by those skilled in the art that in someembodiments of the disclosure the group “-E-” in functional groups -E-MHpresented in the following schemes is equivalent to the group -A- in thedefinition of the composition of a chemotherapeutic moiety as describedherein.

Referring to Scheme 1, in certain embodiments R²⁰ in compounds ofFormula (A) is a protected carboxyl group such as a lower alkyl ester ofa carboxyl group, e.g., a methyl, ethyl, or tert-butyl ester, or abenzyl ester derivative, e.g., benzyl, pentamethylbenzyl, or(4-methoxy)benzyl. In certain embodiments, R²⁰ in compounds of Formula(A) is a tert-butyl ester group (CO₂tBu). In certain embodiments, R²⁰ incompounds of Formula (A) is a methyl ester group (CO₂Me).

Referring to Scheme 1, in certain embodiments, R²⁰ in compounds ofFormula (A) is a protected phosphinic acid derivative, e.g.,1,1-diethyloxyethylethoxyphosphino-1-one (—P(═O)(OEt)[C(OEt)₂Me] (U.S.Pat. No. 8,344,028; Baylis, Tetrahedron Lett, 1995, 36(51), 9385-9388;and Burgos-Lepley, et al., Bioorg. Med. Chem. Lett., 2006, 16,2333-2336). In certain embodiments, R²⁰ in compounds of Formula (A) hasalternatively protected phosphonates and phosphinates as described inthe art (Palacios, et al., Chem. Rev., 2005, 105, 899-931; and Lejzak,et al., J. Enzyme Inhibit., 1993, 7(2), 97-103).

Referring to Scheme 1, in certain embodiments, R²⁰ in compounds ofFormula (A) is a protected sulfinic acid precursor derivative, e.g., a2-mercaptobenzothiazole (Carruthers, et al., Bioorg. Med. Chem. Lett,1995, 5, 237-240; Carruthers, et al., Bioorg. Med. Chem. Lett, 1998, 5,3059-3064; and Okawara, et al., Chem. Lett., 1984, 2015; C. E.Burgos-Lepley, et al., Bioorg. Med. Chem. Lett., 2006, 16, 2333-2336).

Referring to Scheme 1, in certain embodiments, R²⁰ in compounds Formula(A) is a unprotected or protected carboxylic acid (bio)isostereincluding a protected or unprotected 1H-tetrazole (Ballatore, et al.,ChemMedChem, 2013, 8(3), 385-395; Bryans, et al., U.S. Pat. No.6,518,289; and Burgos-Lepley, et al., Bioorg. Med. Chem. Lett., 2006,16, 2333-2336).

Referring to Scheme 1, in certain embodiments of compounds of Formula(A) Q is N(H)—PG where PG is a suitable nitrogen protecting group, e.g.,tert-butoxycarbonyl (Boc), allyloxycarbonyl (alloc), benzyloxycarbonyl(Cbz, Z), ethoxycarbonyl, methoxycarbonyl,(R/S)-1-phenyl-ethoxycarbonyl, (R)-1-phenyl-ethoxycarbonyl,(S)-1-phenyl-ethoxycarbonyl, 1-methyl-1-phenyl-ethoxycarbonyl, formyl,acetyl, trifluoroacetyl, benzoyl, triphenylmethyl (trityl),4-methoxyphenyl-diphenylmethyl, or di-(4-methoxyphenyl)-phenylmethyl,and the like. In certain embodiments, PG in compounds of Formula (A) istert-butoxycarbonyl (Boc) and Q is N(H)Boc (N(H)CO₂tBu). In certainembodiments of compounds of Formula (A) PG is benzyloxycarbonyl (Cbz,Z), and Q is N(H)—Cbz (N(H)COOBn). In certain embodiments of compoundsof Formula (A), PG is acetyl and Q is N(H)—Ac (N(H)COMe).

Referring to Scheme 1, in certain embodiments of compounds of Formula(A) Q is N(PG)₂, where PG is a nitrogen protecting group such as animide-type protecting group, e.g., phthalyl or tert-butoxycarbonyl(Boc). In certain embodiments of compounds of Formula (A) PG is phthalyland Q is N(phthalyl). In certain embodiments of compounds of Formula (A)PG is tert-butoxycarbonyl and Q is N(Boc)₂.

Referring to Scheme 1, in certain embodiments of compounds of Formula(A) the protected amine functionality is an imine where Q is N isCR³⁰R³¹ and each of R³⁰ and R³¹ is independently selected from branchedC₁₋₄ alkyl, non-branched C₁₋₄ alkyl, substituted aryl, non-substitutedaryl, substituted heteroaryl, and non-substituted heteroaryl.

Accordingly, the structures presented in the schemes provided by thepresent disclosure are illustrative rather than comprehensive.

Referring to Scheme 2, in certain embodiments R¹ and/or R⁵, R²⁰, E, thelinker L, and the protecting groups PG and Q are defined as describedherein; one of R², R³, and R⁴ in compounds of Formula (C) is -E-NH₂,wherein E is a bond (“—”), an oxygen atom (—O—), a methylene group(—CH₂—), or methylenoxy group (—CH₂—O—), and wherein MH is an aminogroup (—NH₂) so that -E-NH₂ is equivalent to a) a primary aromatic aminogroup (—NH₂, aniline), b) a primary O-aryl hydroxylamino group (—O—NH₂),c) a primary aminomethyl group (—CH₂—NH₂), or a primary O-benzylhydroxylamino group (—CH₂—O—NH₂). Each of the other remaining R², R³,and R⁴ is hydrogen; each R⁷ and each R⁸ is hydrogen. X is a suitableleaving group e.g., chloro (—Cl) or bromo (—Br).

Referring to Scheme 2, conversion of the primary amino group as incompounds of Formula (B) to the N,N-bis-(2-hydroxyethyl) amino group(N,N-bis-(2-hydroxyethylation)) as in compounds of Formula (C) may beaccomplished by reacting compounds of Formula (B) in suitable solventssuch as about 25-75 vol.-% aqueous acetic acid (HOAc), glacial aceticacid, water, tetrahydrofuran (THF), ethanol (EtOH), 1,4-dioxane, ormixtures of any of the foregoing with an excess of ethylene oxide(oxirane) (about 4-20 equivalents) at a temperature of about −20° C. toabout room temperature for about 12-48 hours. Alternatively, thereaction mixture may be heated in a sealed reaction vessel from about80-140° C. for comparable times (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al, J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Feau, et al., Org. Biomolecular Chem., 2009, 7(24),5259-5270; Springer, et al., J. Med. Chem., 1990, 33(2), 677-681;Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3), 216-226; Buss, etal., J. Fluorine Chem., 1986, 34(1), 83-114; Larden and Cheung,Tetrahedron Lett., 1996, 37(42), 7581-7582; Spreitzer and Puschmann,Monatshefte für Chemie, 2007, 138(5), 517-522; Niculesscu-Duvaz, et al.,J. Med. Chem., 2004, 47(10), 2651-2658; Weisz, et al., Bioorg. Med.Chem. Lett., 1995, 5(24), 2985-2988; Thorn, et al., J. Org. Chem, 1975,40(11), 1556-1558; Baraldini, et al., J. Med., Chem., 2000, 53(14),2675-2684; Zheng, et al., Bioorg., Med., Chem., 2010, 18(2), 880-886;Gourdi, et al., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al.,J. Med. Chem., 1987, 30, 542-547; Matharu, et al., Bioorg. Med. Chem.Lett., 2010, 20, 3688-3691; and Kupczyk-Subotkowska, et al., J. DrugTargeting, 1997, 4(6), 359-370).

Referring to Scheme 2, conversion of the primary amino group as incompounds of Formula (B) to the N,N-bis-(2-hydroxyethyl) amino group(N,N-bis-(2-hydroxyethylation)) as in compounds of Formula (C) may beaccomplished by reacting compounds of Formula (B) in suitable solventssuch water with an excess of about 2-5 equivalents of a suitable2-halogeno ethanol derivative, e.g., 2-chloroethanol (ClCH₂CH₂OH) or2-bromoethanol (BrCH₂CH₂OH), and about 2.0 equivalents of a suitableinorganic base such as sodium bicarbonate (NaHCO₃), sodium carbonate(Na₂CO₃), or calcium carbonate (CaCO₃) at about reflux temperature forabout 8-24 hours. Optionally, the reaction may be carried out in thepresence of a catalytic amount (about 10 mol-%) of potassium iodide (KI)(Palmer, et al., J. Med. Chem. 1990, 33(1), 112-121; Coggiola, et al.,Bioorg. Med. Chem. Lett., 2005, 15(15), 3551-3554; Verny and Nicolas, J.Label. Cmpds Radiopharm., 1988, 25(9), 949-955; and Lin, Bioorg. Med.Chem. Lett., 2011, 21(3), 940-943).

Referring to Scheme 3, in certain embodiments electron-deficient arylhalides of Formula (D), activated with strongly electron withdrawingsubstituents for nucleophilic aromatic substitution reactions (S_(N)Ar)at the aryl ring, may be useful starting materials for incorporatingN,N-bis-(2-functionalized) ethyl amino groups as in compounds of Formula(E) where the corresponding N,N-bis-(2-functionalized)ethyl amino groupsare N,N-bis-(2-hydroxyethyl) amino groups. Commonly used leaving groups(—X) for S_(N)Ar-reactions include halogeno, e.g., fluoro (—F), chloro(—Cl), bromo (—Br), with accessory activating groups at the 2- or4-position relative to the leaving group (ortho- or para-positions).Such groups decrease the electron density in the arene ring and increasethe susceptibility to nucleophilic attack and displacement of theleaving group (—X). Examples of activating, stronglyelectron-withdrawing groups (EWG), include trifluoromethyl (—CF₃), cyano(—CN), nitro (—NO₂), amide (—CON(R¹⁰)₂), and formyl (—CHO).

Useful secondary amines for the introduction of theN,N-bis-(2-hydroxyethyl) amino functionality include diethanolamine(HN(CH₂CH₂OH)₂), protected diethanolamine derivatives, e.g.,O-benzylether protected diethanolamine (HN(CH₂CH₂OBn)₂), or precursorsof the putative N,N-bis-(2-hydroxyethyl)amino group, e.g., 3-pyrroline.Employing O-benzylether protected diethanolamine (HN(CH₂CH₂OBn)₂) or3-pyrroline necessitates conversion of the corresponding intermediatesubstitution products to compounds of Formula (E) bearing the targetN,N-bis-(2-hydroxyethyl)amino groups using methods well known in theart.

Referring to Scheme 3, in certain embodiments R¹ and/or R⁵, R¹⁰, R²⁰,the linker L, the protecting group PG, and Q, the electron withdrawinggroup (EWG), the leaving group (—X), and the secondary amine HNR₂ aredefined as described herein; R¹ and/or R⁵ may also represent an electronwithdrawing group (EWG); one or more of R², R³, and R⁴ in compounds ofFormula (G) or of Formula (H) is a suitable leaving group (—X)), one ormore of R², R³, and R⁴ is a electron withdrawing group (EWG) preferablyin 2- or 4-position relative to the leaving group X; each of the otherremaining R², R³, and R⁴ is hydrogen; each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 3, N,N-bis(2-hydroxyethyl)amino derivatives as incompounds of Formula (E) may be prepared through nucleophilic aromaticsubstitution reactions (S_(N)Ar) of aromatic halides of Formula (D)activated by electron withdrawing groups (EWGs), by reaction with anexcess of about 1.5-5 equivalents of the neat amine, e.g.,HN(CH₂CH₂OH)₂, HN(CH₂CH₂OBn)₂, or 3-pyrroline, (weakly basic reactionconditions) or solutions of the secondary amine in polar aproticanhydrous solvents, e.g., anhydrous dimethylsulfoxide (DMSO),N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), acetonitrile(MeCN), 1,4-dioxane, tetrahydrofuran (THF), or mixtures of the foregoingat a temperature from about 80-200° C. (sealed tube), for about 1-12hours to provide N,N-bis(2-hydroxyethyl)amino-functionalized compoundsof Formula (E). The reaction may also be carried out in the presence ofa catalyst, e.g., copper powder (about 10 mol-%) (Atwell, et al., J.Med. Chem., 2007, 50(6), 1197-1212; Palmer, et al., J. Med. Chem., 1994,37, 2175-2184; Palmer, et al., J. Med. Chem., 1992, 35(17), 3214-3222;Palmer, et al., J. Med. Chem, 1990, 33(1), 112-121; Davies, et al., J.Med. Chem. 2005, 48(16), 5321-5328; Jordan, et al., Bioorg. Med. Chem.,2002, 10(8), 2625-2633; Dheyongera, et al., Bioorg. Med. Chem., 2005,13(3), 689-698; Lin, et al., Bioorg. Med. Chem. Lett., 2011, 21(3),940-943; and Ferlin, et al., Bioorg. Med. Chem., 2004, 12(4), 771-777).

Referring to Scheme 3, methods to convert theN,N-bis-(2-benzyloxyethyl)amino group to a N,N-bis-(2-hydroxyethyl)aminogroup include, for example, catalytic hydrogenolysis of the benzyl ethergroups using heterogeneous catalysts, e.g., 5-10% Pd on carbon (Pd/C) orRaney®-Nickel under standard hydrogenation reaction conditions are knownin the art (Vincent and Prunet, Tetrahedron Lett, 2006, 47(24),4075-4077).

Referring to Scheme 3, conversion the 3-pyrroline ring of theN-aryl-3-pyrroline moiety to a N,N-bis-(2-hydroxyethyl)amino group as incompounds of Formula (E) include oxidative cleavage of the C═C-doublewith the Lemieux-Johnson reagent (osmium tetroxide/sodium periodate,OsO₄/NaIO₄) or by ozonolysis with an O₃/O₂-gas mixture. Reductivework-up, e.g., with borane-dimethylsulfide complex (BH₃.Me₂S),triphenylphosphine (Ph₃P), thiourea (C(═S)(NH₂)₂), or zinc dust, yieldsintermediate N,N-bis(2-oxoethyl)amino groups which may subsequently bereduced to the desired N,N-bis-(2-hydroxyethyl)amino group as incompounds of Formula (E) with suitable reducing reagents, e.g.,borane-THF complex (BH₃.THF), or sodium borohydride (NaBH₄), understandard reaction conditions (Palmer and Denny, Synth. Commun., 1987,17(5), 601-610).

In general, the biological activity of nitrogen mustards is based uponthe presence of a N,N-bis(2-chloroethyl) functionality. Thechemotherapeutic and cytotoxic effects are directly associated with thealkylation of DNA due to the strong electrophilic character of theN,N-bis(2-chloroethyl) functionality. Formation of covalent linkagesincluding interstrand crosslinks (ICLs) is highly cytotoxic and involvesthe disruption of fundamental cellular processes including DNAreplication leading to cellular death.

Many methods and reagents for converting primary alcohols to primaryalkyl chlorides including conversion of N,N-bis(2-hydroxyethyl)aminogroups to N,N-bis(2-chloroethyl)amino groups are known in the art. Themost common methods include the use of concentrated hydrochloric acid(HCl) and various inorganic chlorides of sulfur or phosphorus which areused either in neat form or as solutions in inert solvents such aschlorinated hydrocarbons, aromatic hydrocarbons, or polar non-proticsolvents, at room temperature or at elevated temperatures. Other usefulchlorination methods and reagents include, for example, combinations oftriphenyl phosphine and trichloroacetonitrile (Ph₃P/Cl₃CCN),triphenylphosphine dichloride (Ph₃PCl₂) (prepared from Ph₃P and Cl₂),trimethylsilylchloride and bismuth(III) trichloride (Me₃SiCl/BiCl₃),mixtures of Ph₃P and carbon tetrachloride (CCl₄), or methanesulfonylchloride (MeSO₂Cl) in pyridine at elevated temperatures.

Referring to Scheme 4, it will be appreciated by one skilled in the artthat the presence of particular functional or protecting group incompounds of Formula (F) and Formula (G) determines the choice aparticular reagent, method, or reaction condition for thechloro-de-hydroxylation reaction.

Referring to Scheme 4, in certain embodiments R¹ and/or R⁵, R²⁰, thelinker L, E, the protecting groups PG and Q are defined as describedherein; one of R², R³, and R⁴ in compounds of Formula (F) is a-E-N,N-bis(2-hydroxyethyl)amino group (-E-N(CH₂—CH₂—OH)₂); each of theother remaining R², R³, and R⁴ is hydrogen; and each of R⁷ and R⁸ ishydrogen.

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (F) may be reacted with an excess of about 2-15equivalents of thionyl chloride (SOCl₂) either in neat form or as asolution in an anhydrous organic solvent, e.g., dichloromethane (DCM),chloroform (CHCl₃), 1,2-dichloroethane (DCE), benzene, or mixtures ofany of the foregoing at temperatures from about 0° C. (ice bath)-40° C.or heated at reflux for about 0.5-3 hours to provide compounds ofFormula (M) or of Formula (N) (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al., J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3),216-226; Dheyongera, Bioorg. Med. Chem. 2005, 13(3), 689-698; Zheng,Bioorg. Med. Chem. 2010, 18(2), 880-886; Gourdi, J. Med. Chem., 1990,33(4), 1177-1186; and Lin, et al., Bioorg. Med. Chem. Lett., 2011,21(3), 940-943). The reaction may optionally be carried out in thepresence of a catalytic amount of zinc chloride (ZnCl₂) (10 mol-% to 40mol-%) or in the presence of a catalytic amount of N,N-dimethylformamide(DMF) to facilitate the reaction (Squires, et al., J. Org. Chem., 1975,40(1), 134-136; and Abela Medici, et al, J. Chem. Soc., Perkin Trans. 1,1997, (20), 2258-2263).

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (F) may also be reacted with an excess of about2-10 equivalents of phosphorus(V)oxychloride (phosphoryl chloride,POCl₃) either in neat form or as a solution in an anhydrous organicsolvent, e.g., benzene, acetonitrile, pyridine, or mixtures of any ofthe foregoing at a temperature from about 0° C. (ice bath) to about roomtemperature. The reaction mixture may also be heated from about 80° C.to about reflux temperature for about 0.5-6 hours to provide compoundsof Formula (G) (Palmer, et al., J. Med. Chem. 1990, 33(1), 112-121;Feau, et al., Org. Biomolecular Chem., 2009, 7(24), 5259-5270; Valu, etal., J. Med. Chem., 1990, 33(11), 3014-3019; P. G. Baraldini, et al., J.Med., Chem., 2000, 53(14), 2675-2684; Gourdi, et al., J., Med., Chem.,1990, 33(4), 1177-1186; Haines, et al., J. Med. Chem., 1987, 30,542-547; and Matharu, et al., Bioorg. Med. Chem. Lett., 2010, 20,3688-3691).

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (F) may also be reacted with an excess of carbontetrachloride (CCl₄), optionally in an inert solvent, e.g.,dichloromethane (DCM), in the presence of an excess oftriphenylphosphine (Ph₃P) for about 8-24 hours at about room temperatureor at reflux temperature for about 2-6 hours to provide compounds ofFormula (G) (Buss, et al., J. Fluorine Chem., 1986, 34(1), 83-114; andKupczyk-Subotkowska, et al., J. Drug Targeting, 1997, 4(6), 359-370).

Referring to Scheme 4, in some embodiments N,N-bis(2-hydroxyethyl)compounds of Formula (F) may also be reacted with methanesulfonylchloride (MeSO₂Cl, MsCl) in anhydrous pyridine at about room temperatureor at about 70-100° C. for about 1-3 hours to provide compounds ofFormula (G) (Jordan, et al., Bioorg. Med. Chem., 2002, 10(8), 2625-2633;Abela Medici, et al, J. Chem. Soc., Perkin Trans. 1, 1997, (20),2258-2263; Springer, et al., J. Med. Chem., 1990, 33(2), 677-681; andLarden and Cheung, Tetrahedron Lett., 1996, 37(42), 7581-7582).

Referring to Scheme 5, although halides are common leaving groups innucleophilic substitution reactions for synthetic purposes, it is oftenmore convenient to use the corresponding alcohols such as the ones foundin N,N-bis(2-hydroxyethyl)amino groups of compounds of Formula (H).Since OH is usually considered a poor leaving group, unless protonated,conversion of a hydroxy group such as in N,N-bis(2-hydroxyethyl)aminogroups of compounds of Formula (H) into reactive ester groups, mostcommonly sulfonic ester groups, converts the hydroxyl group into afunctional group with a higher susceptibility to be displaced by anincoming nucleophile including halogenide ions. The N,N-bis(2-aryl- or(polyfluoro)alkylsulfonyloxy)amino groups of aryl- or(polyfluoro)alkylsulfonates of Formula (I) and similar sulfonic estersare most frequently prepared from N,N-bis(2-hydroxy)amino groups ofdiols of Formula (H) through reaction with an appropriate aryl- or(polyfluoro)alkyl-sulfonyl chloride or anhydride in the presence of asuitable base, e.g., pyridine (nucleophilic catalyst). Besides aromatic(R⁴⁰ is (substituted) aryl) sulfonic ester groups, aliphatic (R⁴⁰ isalkyl) sulfonic ester groups, and, in particular, (poly)fluorinated (R⁴⁰is poly-F-alkyl) sulfonic ester groups as still more powerful leavinggroups are frequently used for activation.

Referring to Scheme 5, in certain embodiments the R⁴⁰-group in compoundsof Formula (I) or Formula (K) is for example phenyl and the leavinggroup is phenylsulfonyloxy (PhSO₂O), 4-methylphenyl (para-methylphenyl)and the leaving group is tosylate (4-methylphenylsulfonyloxy, TsO),4-bromophenyl (para-bromophenyl) and the leaving group is brosylate(4-bromophenylsulfonyloxy, BsO), or 4-nitrophenyl (para-nitrophenyl) andthe leaving group is nosylate (4-nitrophenylsulfonyloxy, NsO), methyland the leaving group is mesylate (methanesulfonyloxy, MsO),trifluomethyl and the leaving group is triflate(trifluoromethanesulfonyloxy, TfO), nonafluoro-n-butyl and the leavinggroup is nonaflate (nonafluorobutanesulfonyloxy), or2,2,2-trifluoroethyl and the leaving group is tresylate(2,2,2-trifluoroethanesulfonyloxy). In some embodiments, the R⁴⁰-groupof compounds of Formula (I) and Formula (K) is methyl and the leavinggroup is mesylate (methansulfonyloxy, MsO). In some embodiments, theR⁴⁰-group of compounds of Formula (I) and of Formula (K) istrifluoromethyl and the leaving group is triflate(trifluoromethansulfonyloxy, TfO).

Referring to Scheme 5, N-mustard-type halides of Formula (J), Formula(K), and Formula (L) containing either (a) aN,N-bis(2-halogenoethyl)amino group (compounds of Formula (J)), (b) aN-(2-halogenoethyl)amino-, N-(2-halogeno′ethyl)amino-group (compounds ofFormula (L) or mixed halogeno N-mustards), or (c) aN-(2-halogenoethyl)amino, N-(2-aryl- or(polyfluoro)alkylsulfonyloxyethyl)amino groups (compounds of Formula (K)or hybrid halogeno sulfonate N-mustards), may be prepared from thecorresponding esters of sulfonic acid esters of Formula (P) throughreaction with an excess or a near stoichiometric amount of an alkalimetal halide (MX, MX′) in suitable protic or non-protic organic solventat elevated temperature (halo-de-sulfonyloxy substitution)

Referring to Scheme 5, in certain embodiments M in MX or MX′ is analkali metal cation, e.g., lithium (Li⁺) and sodium (Na⁺), X and X′ inMX or MX′ are halide anions, e.g., chloride (Cl⁻), bromide (Br⁻), andiodide (I⁻). MX or MX′ are alkali metal halides, e.g., lithium chloride(LiCl), lithium bromide (LiBr), sodium chloride (NaCl), sodium bromide(NaBr), or sodium iodide (NaI). In certain compounds of Formula (J),Formula (K), and Formula (L), X is a halogeno, e.g., chloro (—Cl), bromo(—Br), or iodo (—I) (Palmer, et al., J. Med. Chem. 1990, 33(1), 112-121;Palmer, et al., J. Med. Chem., 1994, 37, 2175-2184; Palmer, et al., J.Med. Chem., 1996, 39(13), 2518-2528; Davies, et al., J. Med. Chem. 2005,48(16), 5321-5328; Niculesscu-Duvaz, et al., J. Med. Chem., 2004,47(10), 2651-2658; Weisz, et al., Bioorg. Med. Chem. Lett., 1995, 5(24),2985-2988; Thorn, J. Org. Chem, 1975, 40(11), 1556-1558; Lin, et al.,Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943; Gourdi, et al., J. Med.Chem. 1990, 33(4), 1177-1186; Yang, et al., Tetrahedron, 2007, 63(25),5470-5476; Ferlin, et al., Bioorg. Med. Chem., 2004, 12(4), 771-777; andCoggiola, et al., Bioorg. Med. Chem. Lett., 2005, 15(15), 3551-3554).

Referring to Scheme 5, N-(2-halogenoethyl)amino, N-(2-aryl- oralkylsulfonyloxyethyl)amino groups of Formula (K) (hybrid halogenosulfonate N-mustards) may also be prepared from primary alkyl halides ofFormula (J) containing N,N-bis(2-halogenoethyl)amino groups through (a)a halo-de-halogenation (halide exchange reaction) or (b) a metatheticalsulfonyloxy de-halogeno substitution reaction with solubilized silversulfonates AgOSO₂R⁴⁰, wherein R⁴⁰ is defined as described herein undermild conditions in aprotic organic solvents (Emmons and Ferris, J Am.Chem. Soc., 1953, 75(9), 2257).

Referring to Scheme 5, for example in certain embodiments R¹ and/or R⁵,R²⁰, R⁴⁰, X, X′, E, the linker L, the protecting groups PG and Q aredefined as herein; one of R², R³, and R⁴ in compounds of Formula (H) is-E-N(CH₂—CH₂—OH)₂ each of the other remaining R², R³, and R⁴ ishydrogen; and each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 5, in certain embodiments, theN,N-bis(2-hydroxyethyl)amino group of compounds of Formula (H) may beconverted to N,N-bis(2-(polyfluoro)alkyl- or arylsulfonyloxyethyl)aminogroups of compounds of Formula (I) (S-alkoxy-de-chlorination) byreacting diols of Formula (H) with an excess of a suitable(perfluoro)alkyl- or aryl-sulfonyl anhydride (R⁴⁰SO₂)₂O) (about 2.5-5equivalents), e.g., methanesulfonyl anhydride (R⁴⁰ is methyl (Me),(MeSO₂)₂O)), in an inert solvent such anhydrous dichloromethane (DCM) ortetrahydrofuran (THF) or a mixture of any of the foregoing in thepresence of an excess (about 2-10 equivalents) of a suitable base, e.g.,anhydrous triethylamine (Et₃N, TEA) or anhydrous pyridine, at atemperature from about 0° C. to about room temperature for about 0.5-24hours to afford bis-sulfonic acid esters of Formula (I). The reactionmay optionally be carried out in the presence of a catalytic amount(about 20 mol-%) of 4-N,N-(dimethylamino)pyridine (DMAP).

Referring to Scheme 5, in certain embodiments, using comparable reactionconditions with respect to solvents, bases, stoichiometry of reagents,temperature, catalysts, and duration as described for the reaction ofdiols of Formula (H) with (ployfluoro)alkyl- or aryl-sulfonylanhydrides, diols of Formula (H) may also be reacted with a suitablealkyl- or aryl-sulfonyl halides, e.g., methanesulfonyl chloride (mesylchloride, MsCl) (R⁴⁰ is Me), MeSO₂Cl), to provide the desiredbis-sulfonic acid esters of Formula (I).

Referring to Scheme 5, in certain embodimentsN,N-bis(2-(polyfluoro)alkyl- or aryl-sulfonyloxyethyl)amino groups as incompounds of Formula (I) may be converted (halo-de-sulfonyloxysubstitution) to N,N-bis(halogenoethyl)amino groups of compounds ofFormula (J) by reacting bis-sulfonyl esters of Formula (I) with anexcess of a suitable alkali metal halide salt MX, e.g., lithium chloride(LiCl), lithium bromide (LiBr), sodium chloride (NaCl), sodium bromide(NaBr), or sodium iodide (NaI) (4-16 equivalents) in a suitable organicsolvent, e.g., N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMAc), acetone, 2-butanone (methyl ethyl ketone, MEK),3-methyl-2-butanone (isopropyl methyl ketone, MIPK), acetonitrile(MeCN), methanol (MeOH), tetrahydrofuran (THF), ethyl acetate (EtOAc),or a mixture of any of the foregoing, at room temperature or heated toabout 50-150° C. for about 0.5-6 hours to provide compounds of Formula(J).

Referring to Scheme 5, in certain embodiments using comparable reactionconditions with respect to solvents, temperature, and duration asdescribed for the preparation of compounds of Formula (J), the reactionof bis-sulfonyl esters of Formula (I) may also be carried out in thepresence of about one molar equivalent of a suitable alkali metal halidesalt MX, as defined herein, to provide compounds of Formula (K) bearingN-(2-halogenoethyl)-, N-(2-methylsulfonyloxyethyl) amino groups (mixedhalogeno/sulfonylato N-mustards).

Referring to Scheme 5, in some embodiments compounds of Formula (J) maybe converted to mixed halogeno/sulfonylato N-mustards of Formula (K) byreacting N-mustard derivatives of Formula (J) where X is bromo (—Br)with about 1.0 equivalent or slightly less of a suitable soluble silversulfonate salt, e.g., silver mesylate (AgOSO₂Me, AgOMs) in a polarsolvent such as acetonitrile (MeCN) at about reflux temperature toprovide the mixed halogeno/mesylate N-mustard of Formula (K)(methathetical reaction).

Referring to Scheme 5, in certain embodiments, using comparable reactionconditions with respect to solvents, temperature, and duration asdescribed for the preparation of compounds of Formula (J) and of Formula(K), the reaction of bis-halogeno N-mustards of Formula (J) or of mixedhalogeno/mesylate N-mustards of Formula (R) may also be carried out inthe presence of about one molar equivalent of a suitable alkali metalhalide salt MX′, as defined herein, to provide compounds of Formula (L)bearing N-(2-halogenoethyl)-, N-(2-halogeno′ethyl) amino groups (mixedhalogeno N-mustards).

Reductive N-alkylation is a form of amination/alkylation that involvesthe reaction of an amino group with a carbonyl group to an amine in thepresence of a suitable reducing agent via an intermediate imine orprotonated imine. The carbonyl group component is most commonly analdehyde or ketone functionality, the amino group is most commonlyammonia, a primary or secondary aliphatic amino group, or a primary orsecondary aromatic amino group (aniline). For indirect reductiveaminations, the intermediate imine may be isolated and reduced with asuitable reducing agent. For direct reductive aminations, the reactionmay be carried out simultaneously, with the imine formation andreduction occurring concurrently, typically using reducing agents thatare more reactive toward protonated imines than ketones, and that arestable under moderately acidic conditions, e.g., sodium cyanoborohydride(Na(CN)BH₃) or sodium triacetoxyborohydride (NaB(OAc)₃H.

Referring to Scheme 6, the primary amino group of compounds of Formula(M) either in a suitable salt form, e.g., a hydrochloride (HCl) salt(Ar-E-NH₂.HCl) or as a free base (Ar-E-NH₂) may be subjected to areductive N-alkylation reaction using a suitable halocarbonyl compounds(X is F, Cl or, Br) or derivatives thereof, e.g. a dimethyl acetal, andreducing agents as they are well known in the art (Palani, et al., J.Med. Chem., 2005, 48(15), 4746-4749; van Oeveren, Bioorg. Med. Chem.Lett., 2007, 17(6), 1527-1531; Delfourne, et al., Bioorg. Med. Chem.,2004, 12(15), 3987-3994; Delfourne, et al., J. Med. Chem., 2002, 47(17),3765-3771; and M. Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633).

Suitable halocarbonyl compounds include, for example, 2-chloroaceticacid (ClCH₂CO₂H, X is Cl)), 2-chloroacetaldehyde (ClCH₂CHO, X is Cl)),or 2-bromoacetaldehyde dimethylacetal (MeO)₂CHCH₂Br, X is Br),optionally provided as solutions in suitable solvents, e.g., a 50-wt-%solution of 2-chloroacetaldehyde (ClCH₂CHO, X is Cl)) in water.

Referring to Scheme 6, suitable reducing agents for reductiveN-alkylations of primary amino groups such as in compounds of Formula(M) using 2-chloroacetic acid include boranes, preferablyborane-tetrahydrofuran complex (H₃B.THF), and certain alkalimetalborohydrides, e.g., lithium borohydride (LiBH₄) or sodium borohydride(NaBH₄).

Referring to Scheme 6, the reaction is generally carried out in thepresence of organic solvents such as protic solvents, e.g., methanol(MeOH), acetic acid, (HOAc), trifluoroacetic acid (TFA), 85 wt-%phosphoric acid (H₃PO₄), glacial acetic acid (HOAC), 98 wt-% formicacid, or water, or inert organic solvents, e.g., acetonitrile (MeCN),dichloromethane (DCM), tetrahydrofuran (THF), benzene, or equivalentmixtures of any of the foregoing at a temperature from about 0° C. toabout reflux temperature and for about 0.5-18 hours. In embodimentswhere 2-chloroacetaldehyde is used, suitable reducing agents mayinclude, for example, sodium cyanoborohydride (Na(CN)BH₃), sodiumtriacetoxyborohydride (NaB(OAc)₃H, and sodium borohydride (NaBH₄).

Reduction via hydrogenation is can also be employed. Preferredhydrogenation conditions include catalytic hydrogenation, for example,using palladium on carbon (Pd/C) as the catalyst. As the hydrogensource, gaseous hydrogen (H₂-gas) at pressures ranging from aboutatmospheric pressure to about 150 psi, or suitable ammonium salts, e.g.,ammonium hydrogencarbonate (H₄NHCO₃), may be employed. The hydrogenationmay be carried out at ambient temperature.

Referring to Scheme 6, in certain embodiments, R¹ and/or R⁵, R²⁰, E, thelinker L, the halogeno group X, and the protecting group PG and Q aredefined as herein; one of R², R³, and R⁴ in compounds of Formula (M) is-E-NH₂, wherein E is a bond (“—”), an oxygen atom (—O—), a methylenegroup (—CH₂—), or methylenoxy group (—CH₂—O—), and wherein MH is anamino group (—NH₂) so that -E-NH₂ is equivalent to a) a primary aromaticamino group (—NH₂, aniline), b) a primary O-aryl hydroxylamino group(—O—NH₂), c) a primary aminomethyl group (—CH₂—NH₂), or a primaryO-benzyl hydroxylamino group (—CH₂—O—NH₂); each of the other remainingR², R³, and R⁴ is hydrogen; each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 6, in certain embodiments, the primary amino groupof compounds of Formula (M) may be converted toN,N-bis(2-halogenoethyl)amino groups as in compounds of Formula (N) byreacting compounds of Formula (M) with an excess of about 4-10equivalents of a 2-halogenocarbonyl compound, e.g., a 50 wt-% solutionof 2-chloroacetaldehyde in water, and an excess of about 3-8 equivalentsof a suitable reducing agent, e.g., sodium cyanoborohydride (NaB(CN)H₃).In certain embodiments, the reaction may be carried out in mixtures ofmethanol (MeOH) with trifluoroacetic acid (TFA), glacial acetic acid(HOAc), 98 wt-% formic acid (FA), or 85 wt-% phosphoric acid (H₃PO₄).For example, in certain embodiments, 1:1 (v/v), 2:1 (v/v), or 1:2 (v/v)mixtures MeOH/acid and reaction temperatures from about 0-40° C. andreaction times of about 0.5-18 hours are employed to provide protectedN-mustards of Formula (N).

Estramustine (Emcyt®, Estracit®) is an antimicrotubule chemotherapyagent indicated in the US for the palliative treatment of metastaticand/or progressive prostate cancer. It is derivative of estrogen(specifically, estradiol) with a N-mustard-carbamate ester moiety.

Referring to Scheme 7, methods to functionalize alcohols or phenols withcarbamoyl derivatives of secondary amines yielding carbamates as in, forexample, compounds of Formula (Q) wherein M is oxygen (—O—) and G isoxygen (═O) include carbamoyl chlorides or p-nitrophenyl carbamates, andare well known in the art. Likewise, it is well known in the art thatcarbamates as in, for example, compounds of Formula (Q) wherein M isoxygen (—O—) and G is oxygen (═O) are also accessible through activationof alcohols or phenols with suitable formic ester derivatives includingphosgene (COCl₂), triphosgene (bis(trichloromethyl) carbonate (BTC)), or1,1′-carbonyldiimidazole (CDI) followed by reaction with anappropriately functionalized amine such as HN(CH₂—CH₂—R⁹)₂ wherein R⁹ ischloro (—Cl), bromo (—Br), iodo (—I), or (polyfluoro)alkyl- or arylsulfonyloxy (—OSO₂R⁴⁰) or combinations thereof and R⁴⁰ is defined asdescribed herein.

Likewise and referring to Scheme 7, many methods are known in theliterature and are known by those skilled in the art to preparecompounds of Formula (Q) related to carbamates including a)S-thiocarbamates wherein M is sulfur (—S—) and G is oxygen (═O), b)O-thiocarbamates wherein M is oxygen (—O—) and G is sulfur (═S), c)dithiocarbamates wherein M is sulfur (—S—) and G is sulfur (═S), d)ureas wherein M is nitrogen (—NR¹⁰—), and where R¹⁰ is defined asdescribed herein, and G is oxygen (═O), or thioureas wherein M isnitrogen (—NR¹⁰—) and G is sulfur (═S).

Referring to Scheme 7, in certain embodiments a compound of Formula (O)is, for example, a) a phenol wherein E is a bond (“—”) and MH is ahydroxyl group (—OH), b) an aniline wherein E is a bond (“—”) and MH isan amino group (—NR¹⁰H), c) a thiophenol wherein E is a bond (“—”) andMH is a sulfhydryl group (—SH), d) an O-aryl hydroxylamine wherein E isoxygen (—O—) and MH is an amino group (—NR¹⁰H), e) a benzylic alcoholwherein E is methylene (—CH₂—) and MH is a hydroxyl group (—OH), f) abenzylic amine wherein E is methylene (—CH₂—) and MH is an amino group(—NR¹⁰H), g) a benzylic thiol wherein E is methylene (—CH₂—) and MH issulfhydryl (—SH), h) an O-benzylic hydroxylamine wherein E ismethyleneoxy (—CH₂—O—) and MH is an amino group (—NR¹⁰H).

Referring to Scheme 7, in certain embodiments, R¹ and/or R⁵, R¹⁰, R²⁰,E, M, Z, the linker L, and the protecting group PG and Q are defined asdescribed herein; one of R², R³, and R⁴ in compounds of Formula (O) is-E-MH as described herein; each of the other remaining R², R³, and R⁴ ishydrogen; each of R⁷ and R⁸ is hydrogen; LG is a suitable leaving groupsuch as chloro (—Cl), 4-nitrophenyloxy (NO₂C₆H₄O—), or imidazole; and R⁹is chloro (—Cl), bromo (—Br), iodo (—I), or (polyfluoro)alkyl- or arylsulfonyloxy (—OSO₂R⁴⁰) or combinations thereof, and R⁴⁰ is defined asdescribed herein.

Referring to Scheme 7, in certain embodiments the alcohol, the thiolgroup, or the amino group of compounds of Formula (O) may be convertedto the N,N-bis(2-halogeno- or 2-sulfonyloxyethyl)carbamoyl orN,N-bis(2-halogeno- or 2-sulfonyloxyethyl)thiocarbamoyl group ofcompounds of Formula (Q) by reacting a compound of Formula (O) with, forexample, commercial N,N-bis(2-chloroethyl)carbamoyl chloride (Fex, etal., U.S. Pat. No. 3,299,104), wherein LG is chloro (—Cl), R⁹ is chloro(—Cl), and G is oxygen (═O) or known (4-nitrophenyl)N,N-bis(2-chloroethyl)carbamate where LG is 4-nitrophenol (4-NO₂-Ph-O—),R⁹ is chloro (—Cl), and G is oxygen (═O) in suitable solvents such aspyridine, or triethylamine in 1,4-dioxane/benzene mixtures and the likeat temperatures of about 0-60° C. to provide carbamate, thiocarbamate,or urea derivatives of Formula (Q).

Referring to Scheme 7, in certain embodiments the MH-group of compoundsof Formula (O) may be activated to their corresponding chloroformates,thiochloroformates, or carbonyl imidazoles of Formula (P) with, forexample, phosgene, thiosphosgene, triphosgene, carbonyldiimidazole(CDI), thiocarbonyldiimidazole (TCDI), or the like, in the presence of asuitable base such as inorganic metal-carbonate, e.g., potassiumcarbonate (K₂CO₃) and bicarbonates, e.g., sodium hydrogencarbonate(NaHCO₃), in suitable inert solvents known in the art. Thechloroformates or thiochloroformates of Formula (P) are subsequentlyconverted to the corresponding carbamates of Formula (Q) throughreaction with an appropriately functionalized amine such asHN(CH₂—CH₂—R⁹)₂ wherein R⁹ is chloro (—Cl), bromo (—Br), iodo (—I), or(polyfluoro)alkyl- or aryl sulfonyloxy (—OSO₂R⁴⁰) or combinationsthereof, and R⁴⁰ is defined as described herein, e.g., commercialbis(2-chloroethyl)amine hydrochloride wherein R⁹ is chloro (—Cl) or2-bromo-N-(2-bromoethyl)ethanamine wherein R⁹ is bromo (—Br), and in thepresence of a base such as inorganic metal-carbonate, e.g., potassiumcarbonate (K₂CO₃) and bicarbonate, e.g., sodium hydrogencarbonate(NaHCO₃), ethyl acetate (EtOAc), water, or mixtures of any of theforegoing to yield carbamates of Formula (Q).

In general, the biological activity of nitrogen mustards is based uponthe presence of an alkylating N,N-bis(2-chloroethyl) functionality. Thechemotherapeutic and cytotoxic effects are directly associated with thealkylation of DNA due to the strong electrophilic character of theN,N-bis(2-chloroethyl) functionality. Formation of covalent linkagesincluding interstrand crosslinks (ICLs) is highly cytotoxic and involvesthe disruption of fundamental cellular processes including DNAreplication leading to cellular death.

Because of this property, the nitrogen mustards have been used for anumber of years in laboratory investigations and in the clinical treatfor malignant growth. Unfortunately, the effective dose of nitrogenmustards is in many cases close to the toxic dose and it is thereforedesirable to find a nitrogen mustard or a class of nitrogen mustard typecompounds possessing the high carcinolytic activity of the parentcompound but having modulated toxicity.

The amide linkage masks the alkylating and toxic properties of thenitrogen mustard moiety so that the total host is not subjected toundesirable toxic effects sometime encountered with nitrogen mustardtherapy: the amino acid moiety of the molecule facilitates the selectivedelivery of the “masked” nitrogen mustard via the amino acid transportmechanism into the tumor cells, where the higher amidase activity of thetumor cell liberates the reactivated nitrogen mustard within itself.Thus in effect it will be possible to obtain maximum effect of thenitrogen mustard on the tumor and minimum toxic effect on the host (U.S.Pat. No. 3,235,594).

Referring to Scheme 8, the amide nitrogen mustards of the presentdisclosure are prepared by condensing carboxylic acids of Formula (R)wherein E is a carbonyl group (—C(═O)—) or a methylenecarbonyl group(—CH₂—C(═O)—) with an appropriately functionalized amine such asHN(CH₂—CH₂—R⁹)₂ wherein X is chloro (—Cl), bromo (—Br), iodo (—I), or(polyfluoro)alkyl- or aryl sulfonyloxy (—OSO₂R⁴⁰) or combinationsthereof, and R⁴⁰ is defined as described herein, to provide amides ofnitrogen mustards of Formula (S).

Referring to Scheme 8, a myriad of coupling methods is known in the artto facilitate the formation of amide bonds as in compounds of Formula(S) from carboxylic acids of Formula (R) (Montalbetti and Falque,Tetrahedron, 2005, 61, 10827-10852; and Valeur and Bradley, Chem. Soc.Rev., 2009, 38, 606-631).

Referring to Scheme 8, in certain embodiments, R¹ and/or R⁵, R²⁰, E, thelinker L, and the protecting group PG and Q are defined as describedherein; one of R², R³, and R⁴ in compounds of Formula (R) is -E-OH asdescribed herein; each of the other remaining R², R³, and R⁴ ishydrogen; each of R⁷ and R⁸ is hydrogen; and R⁹ is a suitablefunctionalization providing the alkylation properties of the nitrogenmustard.

Referring to Scheme 8, in certain embodiments the (thio)carboxyl groupof compounds of Formula (R) may be activated as acyl halides, acylazides, symmetrical or unsymmetrical carboxylic, carbonic, or boronicanhydrides, acyl imidazoles, activated esters, phosphonium salts,uronium salts, or ammonium salts followed by ammonolysis of theactivated intermediate either after prior isolation or in situ with anappropriately functionalized amine such as HN(CH₂—CH₂—R⁹)₂ to providenitrogen mustard amides of Formula (S).

Referring to Scheme 9, in certain embodiments the connector group “A” ofthe moiety -A-N(CH₂—CH₂—R⁹)₂ is a bond (“—”), oxygen (—O—), sulfur(—S—), amino (—NR¹⁰—) methylene (—CH₂—), methyleneoxy (—CH₂—O—),oxycarbonyl (—O—C(═O)—), thiocarbonyl (—S—C(═O)—), aminocarbonyl(—NR¹⁰—C(═O)—), oxythiocarbonyl (—O—C(═S)—), thiothiocarbonyl(—S—C(═S)—), aminothiocarbonyl (—NR¹⁰—C(═S)—), methyleneoxycarbonyl(—CH₂—O—C(═O)—), methylenethiocarbonyl (—CH₂—S—C(═O)—),methyleneaminocarbonyl (—CH₂—NR¹⁰—C(═O)—), methyleneoxythiocarbonyl(—CH₂—O—C(═S)—), methylenethiothiocarbonyl (—CH₂—S—C(═S)—),methyleneaminothiocarbonyl (—CH₂—NR¹⁰—C(═S)—), carbonyl (—C(═O)—),methylencarbonyl (—CH₂—C(═O)—), thiocarbonyl (—C(═S)—), ormethylenthiocarbonyl (—CH₂—C(═S)—).

Referring to Scheme 9, in certain embodiments liberation of unprotectedN-mustard functionalized β-substituted β-amino acid derivatives orunprotected N-mustard functionalized β-substituted β-amino acid analogsor carboxylic acid (bio)isosteres of Formula (U) from theircorresponding precursors of Formula (T) may be conducted under aqueousacidic conditions (hydrolysis) (Taylor, et al., Chem. Biol. Drug Des.,2007, 70(3), 216-226; Buss, et al., J. Fluorine Chem., 1986, 34(1),83-114; A. J. Abela, et al, J. Chem. Soc., Perkin Trans. 1, 1997, (20),2258-2263; Weisz, et al., Bioorg. Med. Chem. Lett., 1995, 5(24),2985-2988; Zheng, Bioorg., Med., Chem., 2010, 18(2), 880-886; Haines, etal., J. Med. Chem., 1987, 30, 542-547; and Matharu, et al., Bioorg.,Med., Chem., Lett., 2010, 20, 3688-3691).

Referring to Scheme 9, in certain embodiments liberation of unprotectedN-mustard functionalized β-substituted β-amino acid derivatives orunprotected N-mustard functionalized β-substituted β-amino acid analogsor carboxylic acid (bio)isosteres of Formula (U) from theircorresponding precursors of Formula (T) may also be conducted underanhydrous acidic conditions (Springer, et al., J. Med. Chem., 1990,33(2), 677-681; Davies, et al., J. Med. Chem. 2005, 48(16), 5321-5328;Niculesscu-Duvaz, et al., J. Med. Chem., 2004, 47(10), 2651-2658; Vernyand Nicolas, J. Label. Cmpds, Radiopharm., 1988, 25(9), 949-955; Thorn,et al., J. Org. Chem, 1975, 40(11), 1556-1558; Baraldini, et al., J.Med. Chem., 2000, 53(14), 2675-2684; Gourdi, et al., J. Med. Chem.,1990, 33(4), 1177-1186; and Kupczyk-Subotkowska, et al., J. DrugTargeting, 1997, 4(6), 359-370).

Referring to Scheme 9, it will be understood by those skilled in the artthat protected N-mustard functionalized β-substituted β-amino acidprecursors of Formula (T) or protected N-mustard β-substituted β-aminoacid analog or carboxylic acid (bio)isosteres precursors of Formula (T)bearing different combinations of suitable protecting groups may also beprepared. Different combinations of protecting groups may requirespecific reactants and reaction conditions for effective removal ofspecific set of different protection groups to provide unprotectedN-mustard β-substituted β-amino acid derivatives or unprotectedN-mustard functionalized β-substituted β-amino acid derivatives,analogs, or carboxylic acid (bio)isosteres of Formula (U).

Referring to Scheme 9, in certain embodiments of compounds of Formula(T) and of Formula (U) R¹ and/or R⁵, R⁹, the connector group A, theprotecting groups PG and Q, and the linker L are defined as describedherein; R⁶ is an unprotected carboxylic acid, a carboxylic acid analogor a carboxylic acid (bio)isostere as defined herein; R²⁰ is a protectedcarboxylic acid, a carboxylic acid analog or a carboxylic acid(bio)isostere as defined herein; one of R², R³, and R⁴ is aN,N-bis-(2-functionalized)ethylamino group (nitrogen mustard group)linked to a connector A (-A-N(CH₂—CH₂—R⁹)₂); each of the remaining R²,R³, and R⁴ is hydrogen; each of R⁷ and R⁸ is hydrogen.

Referring to Scheme 9, hydrolytic acidic global deprotection ofcompounds of Formula (T) to provide N-mustard functionalizedβ-substituted β-amino acid derivatives or N-mustard functionalizedβ-substituted β-amino acid analogs or carboxylic acid (bio)isosteres ofFormula (U) may be accomplished by treating protected precursors ofFormula (T) at elevated temperatures from about 40-150° C. with aqueousmineral acids, e.g., 2 M to ˜12 M hydrochloric acid (HCl) for about 6-24hours. In certain embodiments, mixtures of the mineral acid with organicsolvents may be used. A useful aqueous mineral acid reaction mixture tofacilitate global deprotection is, e.g., a 1:1 (v/v) mixture ofconcentrated hydrochloric acid (˜12 M or ˜37 wt-% HCl) with 1,4-dioxane.

Referring to Scheme 9, other aqueous mineral acids with anon-nucleophilic anion known in the art can be used to facilitatehydrolytic acidic global deprotection of compounds of Formula (T)bearing acid-labile or hydrolysis sensitive protecting groups of theprotected carboxylic moiety, of the protected carboxylic acid(bio)isostere, or of the amino functionality of compounds of Formula (T)to provide N-mustard functionalized β-substituted β-amino acidderivatives or N-mustard functionalized β-substituted β-amino acidanalogs or carboxylic acid (bio)isosteres of Formula (U).

Referring to Scheme 9, suitable mineral acids may for example includediluted or concentrated aqueous solutions of hydrobromic acid (HBr),hydroiodic acid (HI), sulfuric acid (H₂SO₄), perchloric acid (HClO₄),and phosphoric acid (H₃PO₄), mixtures of any of the foregoing ormixtures with suitable organic solvents, e.g., 1,4-dioxane, with any ofthe foregoing.

It is within the ability of one skilled in the art to select specificand suitable aqueous mineral acids and reaction conditions forhydrolytic acidic hydrolytic acidic global deprotection of compounds ofFormula (T) to provide N-mustard functionalized β-substituted β-aminoacid derivatives or N-mustard functionalized β-substituted β-amino acidanalogs or carboxylic acid (bio)isosteres of Formula (U).

Referring to Scheme 9, simultaneous global deprotection of compounds ofFormula (T) where R²⁰ is an acid labile moiety derived from a carboxylicacid, e.g., CO₂tBu, CO₂-pentamethylbenzyl, CO₂-(4-methoxy)benzyl, orCO₂-trityl, and Q is a protected amino group derived from an acid-labileN-protecting group, e.g., N(H)Boc, N(H)trityl,N(H)(4-methoxy)phenyl-diphenylmethyl, orN(H)di-((4-methoxy)phenyl)-phenylmethyl, may also be accomplished byreaction with strong organic acids under anhydrous conditions toliberate free (unprotected)N-mustard functionalized β-substitutedβ-amino acid derivatives or N-mustard functionalized β-substitutedβ-amino acid analogs or carboxylic acid (bio)isosteres of Formula (U).

In certain embodiments, strong (organic) acids useful for globaldeprotection under anhydrous conditions include trifluoroacetic acid(TFA), 98 wt-% formic acid (FA), methanesulfonic acid (MeSO₃H), 85 wt-%phosphoric acid (H₃PO₄), 2 M hydrogen chloride (HCl) in diethyl ether(Et₂O), 4 M hydrogen chloride (HCl) in 1,4-dioxane, or a saturatedsolution of HCl in ethyl acetate (EtOAc) (Li, et al., J. Org. Chem.,2006, 71, 9045-9050).

Depending of the overall sensitivity to strong (organic acids),compounds of Formula (T) may be reacted with neat either neat strong(organic) acid or with solutions of the strong organic acid in suitableinert solvents such as dichloromethane (DCM), dichloroethane (DCE),1,4-dioxane, diethylether (Et₂O), tetrahydrofuran (THF), or toluenetypically in ratios ranging from neat (organic) acid to about 10 vol-%(organic) acid in said inert solvent, and reaction temperatures rangingfrom about 0-50° C. for about 1-24 hours to provide unprotectedN-mustard functionalized β-substituted β-amino acid derivatives orunprotected N-mustard functionalized β-substituted β-amino acid analogsor carboxylic acid (bio)isosteres of Formula (U).

Optionally, 2-5 equivalents of a suitable scavenging agent such astriethysilane (Et₃SiH) (TES), triisopropylsilane (iPr₃SiH), thioanisole,or 1,2-dithioethane (HSCH₂CH₂HS) may be added to the reaction mixture tosuppress formation of unwanted side reactions and by-productsoriginating, for example, from alkylation of electron-rich aromaticscaffolds or sulfide groups under global deprotection conditionsdisclosed herein to provide unprotected N-mustard functionalizedβ-substituted β-amino acid derivatives or unprotected N-mustardfunctionalized β-substituted β-amino acid analogs or carboxylic acid(bio)isosteres of Formula (U).

Separation of unprotected N-mustard functionalized β-substituted β-aminoacid derivatives or unprotected N-mustard functionalized β-substitutedβ-amino acid analogs or carboxylic acid (bio)isosteres of Formula (U)from unreacted starting materials, unwanted byproducts, and impuritiesmay be accomplished using, for example, solid-phase extraction (SPE)techniques, e.g., with QMA® cartridges (Waters, USA), LiChrolut®cartridges (EMD Chemicals, USA), or Whatman SAX cartridges (Whatman,USA), preparative normal or reverse phase TLC, reverse phase (RP)semi-preparative or preparative HPLC, crystallization, precipitation, orany other suitable method known in the art.

Purified unprotected N-mustard functionalized β-substituted β amino acidderivatives or unprotected N-mustard functionalized β-substitutedβ-amino acid analogs or carboxylic acid (bio)isosteres of Formula (U)may be isolated using any of the methods known in the art. For example,such methods include removal of HPLC solvents (mobile phase) of thecombined fractions containing the N-mustard functionalized β-substitutedβ-amino acid derivatives or N-mustard functionalized β-substitutedβ-amino acid analogs or carboxylic acid (bioisosteres) of Formula (U)under reduced pressure with a rotary evaporator, or removal of (aqueous)solvent mixtures by primary lyophilization.

Any method known in the art may be used to produce acid addition saltsor salts including pharmaceutically acceptable acid addition salts orsalts of compounds of Formula (U) (Handbook of PharmaceuticalSalts—Properties, Selection, and Use, Stahl and Wermuth, Wiley-VCH,Weinheim, Germany, 2008).

The lyophilization may optionally be conducted in the presence of one ormore equivalents of a mineral acid, optionally with a pharmaceuticallyacceptable counterion, to form (pharmaceutically acceptable) acidaddition salts of compounds of Formula (U). For example, one or moreequivalents of hydrochloric acid (HCl) may be added prior tolyophiliation to form mono-, di-, or polyhydrochloride salts ofcompounds of Formula (U) or mixtures thereof.

The lyophilization may optionally be conducted in the presence of one ormore equivalents of a base, optionally with a pharmaceuticallyacceptable counterion, to form (pharmaceutically acceptable) salts ofcompounds of Formula (U). For example, one or more equivalents of sodiumhydrogen carbonate (NaHCO₃) may be added prior to lyophiliation to formmono-, di-, or poly sodium salts of compounds of Formula (U) or mixturesthereof.

A characteristic feature of solid tumours is the presence of cells atvery low oxygen concentrations (hypoxia; partial pressure of oxygen intumorous tissue of 0.05-5.0%) often surrounding areas of necrosis. Thereare clear links between hypoxia and the lack of response to radiotherapyand intrinsic resistance to cytotoxic therapy. It has also beendemonstrated that hypoxia in tumours tends to select for a moremalignant phenotype (Wilson and Hay, Nat. Rev. Canc., 2011, 11, 393-410;and Brown and Wilson, Nat. Rev. Canc., 2004, 4, 437-447).

Reductive metabolic processes are more prevalent in the hypoxicenvironment of solid tumors. Reductive enzyme systems have the abilityto reduce certain functional groups. For example, aromatic and aliphaticN-oxides (—N⁺(O—)R²) are known to be reducible to the correspondingamines (—NR₂), and nitro groups (—NO₂) can be either reduced to thecorresponding amines (—NH₂) or to hydroxylamines (—NH(OH) depending onthe oxygen saturation of the tissue (Denny, et al., Br. J. Canc., 1996,74, Suppl. XXVII, S32-S38; and Nagasawa, et al., Biol. Pharm. Bull.,2006, 29(12), 2335-2342).

One promising approach for the design of cancer-cell-selective mustardsexploits selective enzymatic reduction of nitroaryl compounds in theoxygen-starved (hypoxic) cells found in solid tumors. N-Oxidederivatives of nitrogen mustards including N-oxides of melphalan(PX-478; Kirkpatrick, et al., U.S. Pat. No. 7,399,785; Koh, et al., Mol.Canc. Ther., 2008, 7(1), 90-100; www.medkoo.com) and chlorambucil(Kirkatrick, et al., Anti-Cancer Drugs, 1994, 5, 467-472; Tercel, etal., J. Med. Chem., 1995, 38, 1247-1252; and Kirckpatrick, U.S. Pat. No.5,602,273) have been investigated as bioreductive prodrugs with reducedsystemic toxicity in comparison to the parent drugs. Those drugs takeadvantage of a) the hypoxic nature, and b) the reductive nature, ofcertain tumorous cells. The N-oxide functional group deactivates theextremely reactive alkylating agent through capture of the lone electronpair of the parent nitrogen mustard moiety thus diminishing thealkylating properties and the off-target toxicities associated withthat. Bioreductive activation within the hypoxic tumor environment ormilieu by hypoxic cells and their reductive enzyme systems is believedto restore the cytotoxicity of the free nitrogen mustards. The overalleffect is an enhanced therapeutic index of the N-oxides of nitrogenmustards relative to their parent nitrogen mustards.

Depending on the pH and the nature of the solvent, particularly aproticorganic solvents, N-oxides of nitrogen mustards are known tointramolecularly rearrange to the corresponding more stablehydroxylamines with markedly less intrinsic cytotoxic potential (Tercel,et al., J. Med. Chem., 1995, 38, 1247-1252; and Kirckpatrick, U.S. Pat.No. 5,602,273). However, it is also known that said hydroxylamines areable to convert back to the parent N-oxides in vivo where the latter canbe reduced in the hypoxic and reductive environment of tumorous cellswhere the underlying nitrogen mustards exerts their cytoxicity.

Referring to Scheme 10, in certain embodiments of compounds of Formula(V), Formula (W), and of Formula (X) R¹ and/or R⁵, R⁶, R⁹, and thelinker L are defined as described herein; one of R², R³, and R⁴ is aN,N-bis-(2-functionalized)ethylamino group (nitrogen mustard group)linked to a connector group “A” (-A-N(CH₂—CH₂—R⁹)₂) wherein theconnector group “A” is a bond (“—”) or a methylene group (—CH₂—); eachof the remaining R², R³, and R⁴ is hydrogen; each of R⁷ and R⁸ ishydrogen.

Referring to Scheme 10, N-oxidation of the N-mustard group of compoundsof Formula (V) with a slight excess of 3-chloroperbezoic acid(meta-chloroperbenzoic acid, mCPBA) in a solvent such as dichloromethane(DCM) at about room temperature followed by work-up with aqueous sodiumhydrogencarbonate furnishes the more stable hydroxylamine (throughputative re-arrangement via a cyclic oxazetidinium species) of Formula(W).

Referring to Scheme 10, N-oxidation of the N-mustard group of compoundsof Formula (V) with 3-5 equivalents of peracetic acid (MeCO(O₂H)),prepared from 35 wt-% aqueous hydrogen peroxide (H₂O₂) in glacial aceticacid (HOAc), in a solvent such as dichloromethane (DCM) at about roomtemperature followed by acid extraction furnishes the correspondingN-oxide of Formula (X).

Characterization

To determine the extent to which compounds provided by the presentdisclosure enter cells via the LAT1/4F2hc transporter, amino acid uptakeassays into cells that are transfected with DNA encoding the LAT1 and4F2hc subunits may be performed using, for example, HEK (human embryonickidney) or CHO (Chinese hamster ovary) cells. Oocytes may also beinjected with cRNA LAT1 and 4F2hc to express LAT1/4F2hc transporter.Compounds may be screened either for specificity for the LAT1/4F2hctransporter or for transport into cells endogenously expressing aplurality of transporters. The results of a screening method (e.g., acompetition uptake, exchange or direct uptake assay) using a cellexpressing the LAT1/4F2hc transporter may be compared with the resultsof a control cell(s) lacking the LAT1/4F2hc transporter or in thepresence of a specific inhibitor of the LAT1/4F2hc transporter.

In competition experiments, the ability of a compound to specificallybind to the LAT1/4F2hc transporter is determined. A known substrate(reference substrate) for the LAT1/4F2hc transporter and a test compoundare added to cells expressing the LAT1/4F2hc transporter. For example,gabapentin may be used as a reference because it demonstrates highselectivity for LAT1/4F2hc. Gabapentin is not a substrate for theintestinal amino acid transporters B^(0,+), ATB⁰⁺, and LAT2, whereasgabapentin may be a substrate for the organic cation transporter OCTN2(Cundy, et al., J Pharm Exp Ther, 2004, 311(1), 315-323; and Grigat, etal., Drug Metabol Disp, 2009, 37(2), 330-337). The amount or rate oftransport of the reference substrate in the presence of the testcompound is compared to the amount or rate of transport of the referencesubstrate in the absence of the test compound. If the amount or rate oftransport of the reference substrate is decreased by the presence of thetest compound, the test compound binds to the LAT1/4F2hc transporter.

Compounds that bind the LAT1/4F2hc transporter can be further analyzedto determine if they are transported by the LAT1/4F2hc transporter oronly compete for binding to the transporter. Transport of a compoundinto a cell can be determined by detecting a signal from within a cellfrom any of a variety of reporters. The reporter can be as simple as alabel such as a fluorophore, a chromophore, a radionuclide, or areporter can be an agent that is detected utilizing liquidchromatography-mass spectroscopy (LC/MS/MS). The same methods ofdetection can be used to determine if a reporter is transported from theintracellular space to the medium by administering the test compound tothe outside of the cell and sampling the media for the presence of theintracellular reporter after a predetermined period of time (exchangeassays).

Having determined that a compound is a substrate for LAT1/4F2hc, afurther screen may be performed to determine the selectivity of thecompound toward other membrane transporters. Selectivity refers to theaffinities with which a compound is transported by differenttransporters. In order to demonstrate selectivity for LAT1/4F2hc, acompound may be tested in uptake and/or competition assays for othertransporters. Transporters that could potentially transport LAT1/4F2hcsubstrates include SLC1A4 (ASCT1; NP_003029), SLC1A5 (ASCT2; NP_005619),SLC6A1 (GAT1; NP_003033), SLC6A5 (GlyT2; NP_004202), SLC6A6 (TauT;NP_003034), SLC6A8 (CT1; NP_005620), SLC6A9 (GlyT1; NM_008865), SLC6A11(GAT3; NP_55044), SLC6A12 (BGT1; NP_003035), SLC6A13 (GAT2; NP_057699),SLC6A14 (ATB^(0,+); NP_009162), SLC6A15 (B⁰AT2; NP_001139807), SLC6A17(XT1; NP_001010898), SLC6A18 (B⁰AT3; NP_872438), SLC6A19 (B⁰AT1;NP_001003841), SLC7A6 (y⁺LAT2; NP_001070253), SLC7A7 (y⁺LAT1;NP_001119577), SLC7A8 (LAT2; NP_036376), SLC7A9 (b^(0,+)AT; NP_055085),SCL7A10 (ASC-1; NP_062823), SLC15A1(PepT1; NP_005064), SLC15A2 (PepT2;NP_066568), SLC16A1 (MCT1; NP_003042), SLC16A2 (MCT8; NP_006508),SLC16A10 (TAT1; NP_061063), SLCO₁B1 (OATP1B1; NP_006437), SLCO₁B3(OATP1B3; NP_062818), SLC22A1 (OCT1; NP_003048), SLC22A2 (OCT2;NP_003049), SLC22A4 (OCTN1; NP_003050), SLC22A5 (OCTN2; NP_003051),SLC22A8 (OAT3; NP_004245), SLC36A1 (PAT1; NP_510968), SLC36A1 (PAT1;NP_510968), SLC36A2 (PAT2; NP_861441), SLC38A1 (SNAT1; NP_109599),SLC38A2 (SNAT2; NP_061849), SLC38A3 (SNAT3; NP_006832), SLC38A4 (SNAT4;NP_060488), SLC38A5 (SNAT5; NP_0277053), SLC43A1 (LAT3; NP_003618), andSLC43A2 (LAT4; NP_689559).

Human genes required for functional expression of a transporter ofinterest may be cloned using PCR, fully sequenced, and subcloned intoplasmids that can be used for expression in mammalian cells or Xenopuslaevis oocytes. Unless otherwise noted, all subunits of a transporter ofinterest are co-expressed in each heterologous system described in theexamples. Because many mammalian cell lines exhibit high levels of aminoacid transport activity, expression in Xenopus laevis oocytes can beadvantageous due to the low levels of endogenous amino acid transport.To assess transport function of a specific transporter protein, it canbe desirable to clone the cDNA and express the protein in cells thathave low endogenous transport activity. Competition assays may beperformed with labeled compounds that are optimal substrates (referencesubstrates) for the transporter of interest. Typically, uptake levels ofa test compound are compared to uptake of a reference substrate for thetransporter of interest.

Compounds of Formula (1) are substrates for LAT1/4F2hc and have aV_(max) of at least 10%, 20%, and in certain embodiments, at least 50%that of gabapentin. Concomitantly, the compounds have a low affinitytoward amino acid transporters of system A, system N, system ASC, andthe system L transporter LAT2/4F2hc.

Biodistribution studies with normal and tumor-bearing rats may be usedto determine the disposition of actively transported compounds and theselectivity of substrate accumulation in tissue that expresses theLAT1/4F2hc transporter compared with other tissue. Imaging techniquescan qualitatively and quantitatively elucidate the role of transportproteins in drug disposition, for example, whole body autoradiography(WBA). WBA allows both the visualization and the quantification ofradionuclide-labeled compound levels in a thin section of the wholeanimal. Information obtained using WBA is analogous to data obtainedfrom diagnostic imaging, albeit at a single point in time.

Pharmaceutical Compositions

Compounds of Formula (1) or pharmaceutically acceptable salts thereofmay be incorporated into pharmaceutical compositions to be administeredto a patient by any appropriate route of administration includingintradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, peroral, sublingual, intracerebral,intravaginal, transdermal, rectal, inhalation, or topical. In certainembodiments, pharmaceutical compositions provided by the presentdisclosure are injectable formulations. In certain embodiments,pharmaceutical compositions provided by the present disclosure areinjectable intravenous formulations. In certain embodiments,pharmaceutical compositions provided by the present disclosure are oralformulations. Oral formulations may be oral dosage forms.

Pharmaceutical compositions provided by the present disclosure maycomprise a therapeutically-effective amount of a compound of Formula (1)or a pharmaceutically acceptable salt thereof together with a suitableamount of one or more pharmaceutically acceptable vehicles so as toprovide a composition for proper administration to a patient. Suitablepharmaceutical vehicles and methods of preparing pharmaceuticalcompositions are described in the art.

In certain embodiments, a compound of Formula (1) or a pharmaceuticallyacceptable salt thereof may be administered by intravenous injection.Suitable forms for injection include sterile aqueous solutions ordispersions of a compound of Formula (1). In certain embodiments, acompound may be formulated in a physiological buffer solution. Prior toadministration, a compound of Formula (1) or a pharmaceuticallyacceptable salt thereof may be sterilized by any art recognized thetechnique, including addition of antibacterial or antifungal agents, forexample, paraben, chlorobutanol, phenol, sorbic acid, thimersol, and thelike. In certain embodiments, a compound of Formula (1) or apharmaceutically acceptable salt thereof may be sterilized by filtrationbefore administration to a subject thereby minimizing or eliminating theneed for additional sterilization agents. An injectable dosage of acompound of Formula (1) may include from about 0.01 mL to about 10 mL,from about 0.1 mL to about 10 mL, from about 0.1 mL to about 5 mL, andin certain embodiments, from about 1 mL to about 5 mL.

Pharmaceutical compositions may comprise a therapeutically effectiveamount of one or more compounds of Formula (1), preferably in purifiedform, together with a suitable amount of a pharmaceutically acceptablevehicle, so as to provide a form for proper administration to a patient.When administered to a patient, the compounds and pharmaceuticallyacceptable vehicles are preferably sterile. Water is a preferred vehiclewhen the compound is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions may also be employed as liquidvehicles, particularly for injectable solutions. Suitable pharmaceuticalvehicles also include excipients such as starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Pharmaceuticalcompositions may also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents may be used.

Pharmaceutical compositions comprising a compound may be manufactured bymeans of conventional mixing, dissolving, granulating, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers, diluents;excipients or auxiliaries, which facilitate processing of compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

Pharmaceutical compositions provided by the present disclosure may takethe form of solutions, suspensions, emulsion, or any other form suitablefor use. Examples of suitable pharmaceutical vehicles are described inthe art.

For parenteral administration, compounds of Formula (1) may beincorporated into a solution or suspension. Parenteral administrationrefers to the administration by injection, for instance by intravenous,intracapsular, intrathecal, intrapleural, intratumoral, orintraperitoneal injection or intravesically. In certain embodiments, acompound of Formula (1) is administered intravenously.

A solution or suspension may also comprise at least one of the followingadjuvants: sterile diluents such as water for injection, saline, fixedoils, polyethylene glycols, glycerol, propylene glycol or othersynthetic solvents, antioxidants such as ascorbic acid or sodiumbisulfite, buffers such as acetates, citrates or phosphates, and agentsfor adjustment of the tonicity such as sodium chloride or dextrose. Aparenteral preparation may be enclosed into ampoules, disposablesyringes or multiple dosage vessels made of glass or plastic.

For topical administration, a compound of Formula (1) may be formulatedas a solution, gel, ointment, cream, suspension, etc. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedmay be used in the formulation. Such penetrants are generally known inthe art. Systemic formulations include those designed for administrationby injection, e.g., subcutaneous, intravenous, intramuscular,intrathecal or intraperitoneal injection, as well as those designed fortransdermal, transmucosal, oral or pulmonary administration. Systemicformulations may be made in combination with a further active agent thatimproves mucociliary clearance of airway mucus or reduces mucousviscosity. These active agents include, for example, sodium channelblockers, antibiotics, N-acetyl cysteine, homocysteine, sodium2-mercaptoethane sulfonate (MESNA), and phospholipids.

When a compound is acidic or basic it may be included in any of theabove-described formulations as the free acid or free base, apharmaceutically acceptable salt, a solvate of any of the foregoing, ora hydrate of any of the foregoing. Pharmaceutically acceptable saltssubstantially retain the activity of the free acid or base, may beprepared by reaction with bases or acids, and tend to be more soluble inaqueous and other protic solvents than the corresponding free acid orbase form.

Assessing single patient response to therapy and qualifying a patientfor optimal therapy are among the greatest challenges of modernhealthcare and relate to trends in personalized medicine. The novelβ-substituted β-amino acid derivatives and β-substituted β-amino acidanalogs provided by the present disclosure have a high selectivity forLAT1/4F2hc. Radio-labeled compounds for positron emission tomography(PET) or Single Photon Emission Computed Tomography (SPECT) with thesame selectivity toward LAT1/4F2hc may be used to predict the efficacyof the treatment based on a single-study, case-by-case patient analysisthus excluding subjects that are expected not to benefit from treatment.PET/SPECT scans using radiolabeled LAT1/4F2hc selective substrates, oncecorrelated to the concentration β-substituted β-amino acid derivativesor β-substituted β-amino acid analogs of Formula (1) can provide athree-dimensional distribution map, which can then be used formacroscopic dose calculations.

Accordingly, it is within the capability of those of skill in the art toassay and use the compounds of Formula (1) and/or pharmaceuticalcompositions thereof for therapy.

Therapeutic Dose

A compound of Formula (1) and/or pharmaceutical composition thereof cangenerally be used in an amount effective to achieve the intendedpurpose. For use to treat a disease such as cancer, a compound ofFormula (1) and/or pharmaceutical compositions thereof, may beadministered or applied in a therapeutically effective amount.

The amount of a compound of Formula (1) and/or pharmaceuticalcomposition thereof that will be effective in the treatment of aparticular disorder or condition disclosed herein will depend in part onthe nature of the disorder or condition, and can be determined bystandard clinical techniques known in the art. In addition, in vitro orin vivo assays may optionally be employed to help identify optimaldosage ranges. The amount of a compound of Formula (1) and/orpharmaceutical composition thereof administered will depend on, amongother factors, the subject being treated, the weight of the subject, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

A compound of Formula (1) may be assayed in vitro and in vivo, for thedesired therapeutic activity, prior to use in humans. For example, invitro assays may be used to determine whether administration of aspecific compound or a combination of compounds is preferred. Thecompounds may also be demonstrated to be effective and safe using animalmodel systems.

In certain embodiments, a therapeutically effective dose of a compoundof Formula (1) and/or pharmaceutical composition thereof will providetherapeutic benefit without causing substantial toxicity. Toxicity ofcompounds of Formula (1) and/or pharmaceutical compositions thereof maybe determined using standard pharmaceutical procedures and may bereadily ascertained by the skilled artisan. The dose ratio between toxicand therapeutic effect is the therapeutic index. In certain embodiments,a compound of Formula (1) and/or pharmaceutical composition thereofexhibits a particularly high therapeutic index in treating disease anddisorders. In certain embodiments, a dose of a compound of Formula (1)and/or pharmaceutical composition thereof will be within a range ofcirculating concentrations that include an effective dose with minimaltoxicity.

Kits

A compound of Formula (1), a pharmaceutically acceptable salt thereof,or a pharmaceutical composition of any of the foregoing may be includedin a kit that may be used to administer the compound to a patient fortherapeutic purposes. A kit may include a pharmaceutical compositioncomprising a compound of Formula (1) suitable for administration to apatient and instructions for administering the pharmaceuticalcomposition to the patient. In certain embodiments, a kit for use intreating cancer in a patient comprises a compound of Formula (1) or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablevehicle for administering the compound, and instructions foradministering the compound to a patient.

Instructions supplied with a kit may be printed and/or supplied, forexample, as an electronic-readable medium, a video cassette, anaudiotape, a flash memory device, or may be published on an internet website or distributed to a patient and/or health care provider as anelectronic communication.

Therapeutic Uses

Compounds of Formula (1) may be used for treating cancer in a patient,wherein the cancerous tissue expresses the LAT1/4F2hc. In certainembodiments, the cancerous tissue expressing the LAT1/4F2hc transporteris in the brain of the patient.

Compounds of Formula (1) may be used in the treatment of a wide varietyof neoplasms where elevated LAT1/4F2hc mediated uptake occurs. Compoundsof Formula (1) are particularly useful for treating brain tumors,including metastases of other solid tumors, such as lung or breastcancer, in the brain.

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be administered totreat a cancer known to be treated by an alkylating agent, such as, forexample, melphalan.

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be used to treat,for example, one or more of the following cancers: adult acutelymphoblastic leukemia (all), childhood acute lymphoblastic leukemia(all), childhood acute myeloid leukemia (aml), adult acute myeloidleukemia (aml), childhood adrenocortical carcinoma, a IDs-relatedcancers, a IDs-related lymphoma, anal cancer, appendix cancer,astrocytoma, childhood atypical teratoid/rhabdoid tumor, basal cellcarcinoma (nonmelanoma), extrahepatic bile duct cancer, childhoodbladder cancer, bone cancer, osteosarcoma, malignant fibroushistiocytoma, childhood craniopharyngioma, childhood brain stem glioma,adult brain tumor, childhood brain tumor, childhood brain stem glioma,childhood central nervous system embryonal tumors, childhood cerebellarastrocytoma, brain tumor, cerebral astrocytoma/malignant glioma, ductalcarcinoma in situ, childhood ependymoblastoma, childhood ependymoma,childhood esthesioneuroblastoma, childhood medulloblastoma, childhoodmedulloepithelioma, childhood pineal parenchymal tumors of intermediatedifferentiation, supratentorial primitive neuroectodermal tumors andpineoblastoma, childhood visual pathway and hypothalamic glioma,childhood brain and spinal cord tumors, breast cancer, childhood breastcancer, male breast cancer, childhood bronchial tumors, hematopoetictumors of the lymphoid lineage, hematopoetic tumors of the myeloidlineage, burkitt lymphoma, childhood carcinoid tumor, gastrointestinalcarcinoid tumor, carcinoma of head and neck, childhood central nervoussystem embryonal tumors, primary central nervous system lymphoma,childhood cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,childhood cervical cancer, childhood cancers, childhood chordoma,chronic lymphocytic leukemia (cll), chronic myeloproliferativedisorders, colorectal cancer, cutaneous t-cell lymphoma, childhoodcentral nervous system embryonal tumors, desmoplastic small round celltumor, endometrial cancer, childhood ependymoblastoma, childhoodependymoma, esophageal cancer, childhood esophageal cancer, ewing familyof tumors, childhood extracranial germ cell tumor, extragonadal germcell tumor, extrahepatic bile duct cancer, dye cancer, Intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,childhood gastric (stomach) cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (gIst), childhood gastrointestinalstromal cell tumor, childhood extracranial germ cell tumor, extragonadalgerm cell tumor, ovarian germ cell tumor, gestational trophoblastictumor/disease, adult glioma, glioblastoma, childhood brain stem,childhood cerebral astrocytoma, childhood visual pathway andhypothalamic glioma, hairy cell leukemia, childhood heart cancer, headand neck cancer, childhood head and neck cancer, adult (primary)hepatocellular (liver) cancer, childhood (primary) hepatocellular(liver) cancer, adult Hodgkin lymphoma, childhood Hodgkin lymphoma,hypopharyngeal cancer, childhood hypothalamic and visual pathway glioma,intraocular melanoma, pancreatic neuroendocrine tumors (islet celltumors), endocrine pancreas tumors (islet cell tumors), Kaposi sarcoma,kidney (renal cell) cancer, kidney cancer, laryngeal cancer, childhoodlaryngeal cancer, adult acute lymphoblastic leukemia, childhood acutelymphoblastic leukemia, adult acute myeloid leukemia, childhood acutemyeloid leukemia, chronic myelogenous leukemia (cml), hairy cellleukemia, lip and oral cavity cancer, adult primary liver cancer,childhood primary liver cancer, non-small cell lung cancer, small celllung cancer, a IDs-related lymphoma, Burkitt lymphoma, t-cell lymphoma,b-cell lymphoma, cutaneous t-cell lymphoma, adult Hodgkin lymphoma,childhood Hodgkin lymphoma, adult non-Hodgkin lymphoma, childhoodnon-Hodgkin lymphoma, primary central nervous system lymphoma,langerhans cell histiocytosis, Waldenström macroglobulinemia, malignantfibrous histiocytoma of bone and osteosarcoma, childhoodmedulloblastoma, childhood medulloepithelioma, melanoma, intraocular(dye) melanoma, Merkel cell carcinoma, adult malignant mesothelioma,childhood mesothelioma, primary metastatic squamous neck cancer withoccult, mouth cancer, myelodysplastic/myeloproliferative neoplasms,midline tract carcinoma involving nUt gene, childhood multiple endocrineneoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosisfungoides, myelodysplastic syndromes myelodysplastic/myeloproliferativediseases, chronic myelogenous leukemia, adult acute myeloid leukemia,childhood acute myeloid leukemia, multiple myeloma, chronicmyeloproliferative disorders, malignant germ cell tumors, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, childhoodnasopharyngeal cancer, neuroblastoma, adult non-Hodgkin lymphoma,childhood non-Hodgkin lymphoma, non-small cell lung cancer, childhoodoral cancer, lip and oral cavity cancer, oropharyngeal cancer,osteosarcoma and malignant fibrous histiocytoma of bone, childhoodovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor,ovarian low malignant potential tumor, pancreatic cancer, childhoodpancreatic cancer, islet cell tumors, childhood papillomatosis,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, childhood pinealparenchymal tumors of intermediate differentiation, childhoodpineoblastoma and supratentorial primitive neuroectodermal tumors,pituitary tumor, paraganglioma, plasma cell neoplasm/multiple myeloma,pleuropulmonary blastoma, childhood pleuropulmonary blastoma, primarycentral nervous system (cns) lymphoma, pregnancy and breast cancer,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell (kidney) cancer, childhood renal cell (kidney) cancer, renalpelvis and ureter, transitional cell cancer, respiratory tract carcinomainvolving the nUt gene on chromosome 15, retinoblastoma, childhoodrhabdomyosarcoma, salivary gland cancer, childhood salivary glandcancer, sarcoma (dwing family of tumors), Kaposi sarcoma, adult softtissue sarcoma, childhood soft tissue sarcoma, uterine sarcoma, sézarysyndrome, skin cancer (nonmelanoma), childhood skin cancer, melanoma,Merkel cell skin carcinoma, small cell lung cancer, small intestinecancer, adult soft tissue sarcoma, childhood soft tissue sarcoma,squamous cell carcinoma (nonmelanoma), primary and metastatic squamousneck cancer with occult, stomach (gastric) cancer, childhood stomach(gastric) cancer, childhood supratentorial primitive neuroectodermaltumors, cutaneous t-cell lymphoma, testicular cancer, throat cancer,thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma,thyroid cancer, childhood thyroid cancer, gestational trophoblastictumor, adult unknown primary site, carcinoma of, childhood cancer ofunknown primary site, unusual cancers of childhood, transitional cellcancer of ureter and renal pelvis, urethral cancer, endometrial uterinecancer, uterine sarcoma, vaginal cancer, childhood vaginal cancer,childhood visual pathway and hypothalamic glioma, vulvar cancer,Waldenström macroglobulinemia, Wilms tumor, women's cancers, andsystemic and central metastases of any of the foregoing.

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be used to treat,for example, one or more of the following cancers wherein the cancer isselected from any of the primary adult and childhood brain and CNScancers including glioblastoma (GBM) and astrocystoma, skin cancersincluding melanoma, lung cancers including small cell lung cancers,non-small cell lung cancers (NSCLC), and large cell lung cancers,breasts cancers including triple negative breast cancer (TNBC), bloodcancers including myelodysplastic syndrome (MDS), multiple myeloma (MM),and acute myeloid leukemia (AML), prostate cancer including castrateresistant prostate cancer (CRPC), liver cancers including hepatocellularcarcinoma (HCC), esophageal and gastric cancers, and any systemic andcentral metastases of any of the foregoing.

Compounds of Formula (1) maybe used to treat a cancer in which there isdifferential LAT1/4F2hc transport activity relative to surroundingtissue and/or tissue in other body organs. Patients having a tumorexhibiting a greater LAT1/4F2hc transport activity than non-diseasedtissue are expected to respond more favorably to treatment with atherapeutic agent that is a substrate for the LAT1/4F2hc transporter andto experience fewer adverse effects associated with the effects of thetherapeutic agent on non-diseased tissue. Compounds of Formula (1) aretherapeutic agents, are substrates for the LAT1/4F2hc transporter, andexhibit cytotoxicity.

The amount of a compound of Formula (1) that will be effective in thetreatment of a cancer will depend, at least in part, on the nature ofthe disease, and may be determined by standard clinical techniques knownin the art. In addition, in vitro or in vivo assays may be employed tohelp identify optimal dosing ranges. Dosing regimens and dosingintervals may also be determined by methods known to those skilled inthe art. The amount of compound of Formula (1) administered may dependon, among other factors, the subject being treated, the weight of thesubject, the severity of the disease, the route of administration, andthe judgment of the prescribing physician.

For systemic administration, a therapeutically effective dose may beestimated initially from in vitro assays. Initial doses may also beestimated from in vivo data, e.g., animal models, using techniques thatare known in the art. Such information may be used to more accuratelydetermine useful doses in humans. One having ordinary skill in the artmay optimize administration to humans based on animal data.

A dose of compound of Formula (1) and appropriate dosing intervals maybe selected to maintain a sustained therapeutically effectiveconcentration of the compound of Formula (1) in the blood of a patient,and in certain embodiments, without exceeding a minimum adverseconcentration.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered once per day, twice per day,and in certain embodiments at intervals of more than once per day.Dosing may be provided alone or in combination with other drugs and maycontinue as long as required for effective treatment of the disease.Dosing may also be undertaken using continuous or semi-continuousadministration over a period of time. Dosing includes administering apharmaceutical composition to a mammal, such as a human, in a fed orfasted state.

A pharmaceutical composition may be administered in a single dosage formor in multiple dosage forms or as a continuous or an accumulated doseover a period of time. When multiple dosage forms are used the amount ofcompound of Formula (1) contained within each of the multiple dosageforms may be the same or different.

Suitable daily dosage ranges for administration may range from about 2mg to about 50 mg of a compound of Formula (1) per kilogram body weight.

Suitable daily dosage ranges for administration may range from about 1mg to about 100 mg of a compound of Formula (1) per square meter (m²) ofbody surface.

In certain embodiments, a compound of Formula (1) may be administered totreat cancer in a subject in an amount from about 50 mg to about 2,000mg per day, from about 100 mg to about 1,500 mg per day, from about 200mg to about 1,000 mg per day, or in any other appropriate daily dose.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered to treat cancer in a subjectso as to provide a therapeutically effective concentration of a compoundof Formula (1) in the blood or plasma of the subject. In certainembodiments, a therapeutically effective concentration of a compound ofFormula (1) in the blood or plasma of a subject is from about 1 μg/mL toabout 60 μg/mL, from about 2 μg/mL to about 50 μg/mL, from about 5 μg/mLto about 40 μg/mL, from about 5 μg/mL to about 20 μg/mL, and in certainembodiments, from about 5 μg/mL to about 10 μg/mL. In certainembodiments, a therapeutically effective concentration of a compound ofFormula (1) in the blood or plasma of a subject is at least about 2μg/mL, at least about 5 μg/mL, at least about 10 μg/mL, at least about15 μg/mL, at least about 25 μg/mL, and in certain embodiments, at leastabout 30 μg/mL. In certain embodiments, a therapeutically effectiveconcentration of a compound of Formula (1) in the blood or plasma of asubject is less than an amount that causes unacceptable adverse effectsincluding adverse effects to homeostasis. In certain embodiments, atherapeutically effective concentration of a compound of Formula (1) inthe blood or plasma of a subject is an amount sufficient to restoreand/or maintain homeostasis in the subject.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered to treat cancer in a subjectso as to provide a therapeutically effective concentration of a compoundof Formula (1) in the blood or plasma of a subject for an extendedperiod of time such as, for example, for at least about 4 hours, for atleast about 6 hours, for at least about 8 hours, for at least about 10hours, and in certain embodiments, for at least about 12 hours.

The amount of a compound of Formula (1) administered may vary during atreatment regimen.

Pharmaceutical compositions provided by the present disclosure mayfurther comprise one or more pharmaceutically active compounds inaddition to a compound of Formula (1). Such compounds may be provided totreat the cancer being treated with the compound of Formula (1) or totreat a disease, disorder, or condition other than the cancer beingtreated with the compound of Formula (1).

In certain embodiments, a compound of Formula (1) may be used incombination with at least one other therapeutic agent. In certainembodiments, a compound of Formula (1) may be administered to a patienttogether with another compound for treating cancer in the subject. Incertain embodiments, the at least one other therapeutic agent may be adifferent compound of Formula (1). A compound of Formula (1) and the atleast one other therapeutic agent may act additively or, and in certainembodiments, synergistically. The at least one additional therapeuticagent may be included in the same pharmaceutical composition or vehiclecomprising the compound of Formula (1) or may be in a separatepharmaceutical composition or vehicle. Accordingly, methods provided bythe present disclosure further include, in addition to administering acompound of Formula (1), administering one or more therapeutic agentseffective for treating cancer or a different disease, disorder orcondition than cancer. Methods provided by the present disclosureinclude administration of a compound of Formula (1) and one or moreother therapeutic agents provided that the combined administration doesnot inhibit the therapeutic efficacy of a compound of Formula (1) and/ordoes not produce adverse combination effects.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered concurrently with theadministration of another therapeutic agent, which may be part of thesame pharmaceutical composition as, or in a different pharmaceuticalcomposition than that comprising a compound of Formula (1). A compoundof Formula (1) may be administered prior or subsequent to administrationof another therapeutic agent. In certain embodiments of combinationtherapy, the combination therapy may comprise alternating betweenadministering a compound of Formula (1) and a composition comprisinganother therapeutic agent, e.g., to minimize adverse drug effectsassociated with a particular drug. When a compound of Formula (1) isadministered concurrently with another therapeutic agent thatpotentially may produce an adverse drug effect including, for example,toxicity, the other therapeutic agent may be administered at a dose thatfalls below the threshold at which the adverse drug reaction iselicited.

In certain embodiments, pharmaceutical compositions comprising acompound of Formula (1) may be administered with one or more substancesto enhance, modulate and/or control release, bioavailability,therapeutic efficacy, therapeutic potency, stability, and the like of acompound of Formula (1). For example, to enhance the therapeuticefficacy of a compound of Formula (1), a compound of Formula (1) or apharmaceutical composition comprising a compound of Formula (1) may beco-administered with one or more active agents to increase theabsorption or diffusion of the compound of Formula (1) from thegastrointestinal tract to the systemic circulation, or to inhibitdegradation of the compound of Formula (1) in the blood of a subject. Incertain embodiments, a pharmaceutical composition comprising a compoundof Formula (1) may be co-administered with an active agent havingpharmacological effects that enhance the therapeutic efficacy of thecompound of Formula (1).

In certain embodiments, a compound of Formula (1) or a pharmaceuticalcomposition comprising a compound of Formula (1) may be administered inconjunction with an agent known or believed to be effective in treatingcancer in a patient.

For example, in certain embodiments, a compound of Formula (1) or apharmaceutical composition comprising a compound of Formula (1) may beadministered in conjunction with another chemotherapeutic agents, suchas, for example, N-acetyl cysteine (NAC), adriamycin, alemtuzumab,amifostine, arsenic trioxide, ascorbic acid, bendamustine, bevacizumab,bortezomib, busulfan, buthionine sulfoxime, carfilzomib, carmustine,clofarabine, cyclophosphamide, cyclosporine, cytarabine, dasatinib,datinomycin, defibrotide, dexamethasone, docetaxel, doxorubicin,etoposide, filgrastim, floxuridine, fludarabine, gemcitabine, interferonalpha, ipilimumab, lenalidomide, leucovorin, melphalan, mycofenolatemofetil, paclitaxel, palifermin, panobinostat, pegfilrastim,prednisolone, prednisone, revlimid, rituximab, sirolimus, sodium2-mercaptoethane sulfonate (MESNA), sodium thiosulfate, tacrolimus,temozolomide, thalidomide, thioguanine, thiotepa, topotecan, velcade, ora combination of any of the foregoing. In certain embodiments, acompound of Formula (1) and/or pharmaceutical compositions thereof canbe used in combination therapy with other chemotherapeutic agentsincluding one or more antimetabolites such as folic acid analogs;pyrimidine analogs such as fluorouracil, floxuridine, and cytosinearabinoside; purine analogs such as mercaptopurine, thiogunaine, andpentostatin; natural products such as vinblastine, vincristine,etoposide, tertiposide, dactinomycin, daunorubicin, doxurubicin,bleomycin, mithamycin, mitomycin C, L-asparaginase, and interferonalpha; platinum coordination complexes such as cis-platinum, andcarboplatin; mitoxantrone; hydroxyurea; procarbazine; hormones andantagonists such as prednisone, hydroxyprogesterone caproate,medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone,flutamide, and leuprolide, anti-angiogenesis agents or inhibitors suchas angiostatin, retinoic acids, paclitaxel, estradiol derivatives, andthiazolopyrimidine derivatives; apoptosis prevention agents; andradiation therapy.

In certain embodiments, a compound of Formula (1) may be coadministeredwith a compound that inhibits DNA repair such as, for example,O6-benzylguanine (O6-BG).

In certain embodiments, a compound of Formula (1) may be coadministeredwith a compound that blocks and/or inhibits transporters other than LAT1such as, for example, amino acids. In certain embodiments, compounds ofFormula (1) may be administered to a patient together with one or moreamino acids such as, for example, arginine (Arg), serine (Ser), lysine(Lys), asparagine (Asn), glutamine (Gln), threonine (Thr), or mixturesof any of the foregoing. In certain embodiments, co-administration ofamino acids is intended to saturate amino acid transporters thatinteract with compounds of Formula (1) and thereby increase theselectivity for LAT1.

The efficacy of administering a compound of Formula (1) for treatingcancer may be assessed using in vitro and animal studies and in clinicaltrials.

The suitability of compounds of Formula (1) and/or pharmaceuticalcompositions thereof in treating cancers listed above may be determinedby methods described in the art. For example, screens developed todemonstrate the anti-tumor activity of oncolytic agents are known(Miller, et al., J Med Chem, 1977, 20(3), 409-413; Sweeney, et al.,Cancer Res, 1978, 38(9), 2886-2891; and Weiss and Von Hoff, Semin Oncol,1985, 12(3 Suppl 4), 69-74). Accordingly, it is well with the capabilityof those of skill in the art to assay and use the compounds and/orpharmaceutical compositions thereof to treat the above diseases ordisorders.

Methods provided by the present disclosure have use in animals,including mammals, such as in humans.

EXAMPLES

The following examples describe in detail the synthesis of compounds ofFormula (1), characterization of compounds of Formula (1), and uses ofcompounds of Formula (1). It will be apparent to those skilled in theart that many modifications, both to materials and methods, may bepracticed without departing from the scope of the disclosure.

General Experimental Protocols

All reagents and solvents were purchased from commercial suppliers andused without further purification or manipulation.

Proton NMR spectra were recorded on a Varian Mercury Plus300 MHzSpectrometer equipped with an Oxford magnet, a Sun Sunblade 150 hostcomputer, a Solaris operating system, VNMR data processing software, anda HP LaserJet printer. Where specifically noted, a Varian VNMRS 400Spectrometer was used (400 MHz). CDCl₃ (99.8% D), MeOH-d⁴ (CD₃OD, 99.8+%D), deuteroxide (D₂O) (99.8+% D) were used as recording solvents unlessotherwise noted. The CHCl₃, MeOH-d³, HDO solvent signals ortetramethylsilane (TMS) were used for calibration of the individualspectra.

Analytical thin layer chromatography (TLC) was performed using EMDMillipore aluminum-backed TLC sheets (EMD5554-7) pre-coated with silicagel 60 F254 (200 μm thickness, 60 Å pore size) where F254 is afluorescent indicator with a 254 nm excitation wavelength. An ENF-240CSpectroline® UV-lamp (Spectronics Corporation, USA) was used for TLCdetection and visualization. Dyeing or staining reagents for TLCdetection and visualization, e.g., an ethanolic ninhydrin solution or a0.2 wt-% aqueous potassium permanganate (KMnO₄) solution, were preparedaccording methods known in the art.

Analytical LC/MS was performed on a Shimadzu LC/MS-2020 ProminenceSeries system equipped with CBM-20A communication bus module (Shimadzu228-45012-32), a SPD-20AV UV/VIS detector (Shimadzu 228-45004-32), aSIL-20AC autosampler (Shimadzu 228-45136-32), DGU-20A5 degasser(Shimadzu 228-45019-32), two LC-20AD XP HPLC pumps (Shimadzu228-45137-32), an Agilent Zorbax 5 μm XDB-C18 2.1×50 mm column (Agilent960 967-902), and a commercial desktop computer and printer for datacomputation. Gradients of water (solvent A) (Arrowhead, Nestle NorthAmerica, Inc.) and acetonitrile (MeCN; solvent B) (EMD AX0145-1 orAldrich CHROMASOLV® 439134) containing 0.075 vol-% of formic acid (EMDFX0440-7) were used in analytical LC/MS analyses.

Analytical LC/UV was performed on an Agilent 1100 Series system equippedwith an Agilent 1100 Series degasser (Agilent G1379A), an Agilent 1100Series quad pump (Agilent G1311A), an Agilent 1100 Series autosampler(ALS) (Agilent G1329A), an Agilent 1100 Series COLCOM (Agilent G1316A),a Phenomenex Gemini C18 5 μm 110 Å pore size 150×4.6 mm HPLC column(Phenomenex 00F-4435-E0), a Compaq Presario personal computer, and a HPLaserJet P2015 printer for data computation. Gradients of water (solventA) (Arrowhead, Nestle North America, Inc.) and acetonitrile (MeCN;solvent B) (EMD AX0145-1 or Aldrich CHROMASOLV® 439134) containing 0.075vol-% of formic acid (EMD FX0440-7) were used in analytical LC/UVanalyses.

Preparative HPLC was conducted with a Varian ProStar Series systemequipped with a Model 340 UV-C UV-VIS detector, a Model 210 solventdelivery module, a Hamilton PRP-112-20 μm 100 Å 21.2×250 mm preparativeHPLC column (Hamilton 79428), and a commercial desktop personal computerfor data computation. Gradients of water (solvent A) (Arrowhead, NestleNorth America, Inc.) and acetonitrile (MeCN; solvent B) (EMD AX0145-1 orAldrich CHROMASOLV® 439134) containing 0.1 vol-% of formic acid (EMDFX0440-7) were used for preparative HPLC purifications.

Compound isolation from aqueous solvent mixtures, e.g.,acetonitrile/water/0.1 vol-% formic acid, was accomplished by primarylyophilization of pooled and frozen (after freeze drying) fractionsunder reduced pressure at room temperature using manifold freeze dryerssuch as Heto Drywinner DW 6-85-1, Heto FD4, or VIRTIS Freezemobile 25 ESequipped with a high vacuum pump. Optionally, and if the isolatedcompound had ionizable functional groups such as an amino group or acarboxylic acid, the lyophilization process was conducted in thepresence of an excess (about 1.1 to 5.0 equivalents) of 1.0 Mhydrochloric acid (HCl) to yield the purified compound(s) as thecorresponding hydrochloride salt (HCl-salt), dihydrochloride salts,and/or the corresponding protonated free carboxylic acid. Melting pointswere determined in duplicate with a SRS OptiMelt MPA-100 automatedmelting point system with digital imaging processing technology and areuncorrected (Stanford Research Systems, USA).

Filtrations were conducted using commercial Celite® 545 (EMD CX0574-1)which was compressed in to glass Büchner-funnels to create a plug of 2-5cm thickness. Reaction mixtures containing precipitated reaction sideproducts or heterogenous catalyst residues were filtered off usingstandard techniques. Care must be taken filtering off activatedcatalysts or finely dispersed metals (ignition!).

Unless otherwise noted, aqueous work-up typically constitutes dilutionof a crude reaction product, with or without residual reaction solvent,with 1.0 M hydrochloric acid (HCl) or a saturated aqueous solution ofammonium chloride (NH₄Cl), multiple extraction with an organic solvent,e.g., ethyl acetate (EtOAc), diethyl ether (Et₂O), or dichloromethane(DCM), washing with water, a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃), and brine (saturated aqueous solution ofsodium chloride (NaCl)), drying of the organic phase (combined organicextracts) over anhydrous magnesium sulfate (MgSO₄) (EMD MX0075-1) orsodium sulfate (Na₂SO₄) (EMD SX0760E-3), filtration, washing of thefilter residue, and evaporation of the combined filtrates under reducedpressure using a rotary evaporator at room or elevated temperaturefollowed by compound purification e.g., silica gel columnchromatography, crystallization or titruation.

Silica gel column chromatography was conducted with silica gel (about100-200 mL silica gel per gram of compound) 600.04-0.063 mm (40-63 μm,230-400 mesh) (EMD MilliporeEM1.09385.9026/EM1.09385.1033/EM1.09385.2503) using single solvents ormixtures of suitable solvents, e.g., ethyl acetate (EtOAc) and hexane ordichloromethane (DCM) and methanol (MeOH), as determined by TLC.Samples/fractions containing desired product detected by analytical TLCand/or analytical LC/MS, or LC/UV were pooled and the solvents wereremoved under reduced pressure using a Heidolph Laborota 4001 Efficientrotary evaporator (Heidolph, Germany) (Heidolph 519-10000-01-5) equippedwith a HB digit heating bath (Heidolph 517-01002-01-4), and a Rotavacvalve control vacuum pump (Heidolph 591-00130-01-0).

Chemical names were generated using the ChemDraw Ultra 12.0(CambridgeSoft, Cambridge, Mass., USA) nomenclature program.

Description 1 General Procedure for the Reduction of Benzoic Acids toBenzylic Alcohols

Adapting literature known protocols (Hay, et al., J. Chem. Soc., PerkinTrans. 1, 1999, 2759-2770; Fujikawa, et al., J. Am. Chem. Soc., 2008,130, 14533-14543; Allen, et al., International Publication No. WO2010/122089; and Gerspacher, et al., International Publication No.WO2008/031594), commercial borane dimethylsulfide (BH₃.DMS, BH₃.SMe₂)(2.0 M in THF) (50 mL, 100 mmol) or borane tetrahydrofurane complex(BH₃.THF) (1.0 M in THF) (100 mL, 100 mmol) is added dropwise at roomtemperature to a stirred solution of the nitrobenzoic acid (50 mmol) inanhydrous THF (250 mL). Optionally, the reaction is performed in thepresence of trimethyl borate (B(OMe)₃) (200 mmol). The solution isheated at reflux for 4-6 hours (˜75° C. oil bath temperature). Thereaction is monitored by TLC and/or LCMS to completion. After cooling toabout 5° C. (ice bath), the reaction is carefully quenched with a 1:1(v/v) mixture of methanol (MeOH)/water (25 mL) followed by 5 Nhydrochloric acid (HCl) (50 mL). The mixture is heated at about 50° C.for about 30-60 min and the majority of the volatile solvents areremoved under reduced pressure. Water is added and the aqueous phase isextracted with ethyl acetate (3×). The combined organic extracts aresuccessively washed with a saturated aqueous sodium hydrogencarbonate(NaHCO₃) solution (1×) and with brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and the solvents are evaporated todryness under reduced pressure. If needed, the crude material ispurified by silica gel column chromatography or is re-crystallized.

Description 2 General Procedure for the Oxidation of Benzylic Alcoholsto Aromatic Aldehydes

Variant A:

Adapting literature known protocols (Parikh, et al., J. Am. Chem. Soc.1967, 89, 5505-5507; and Jandeleit, et al., U.S. Pat. No. 8,168,617), toa solution of the alcohol (50 mmol), dimethylsulfoxide (DMSO) (28.5 mL,400 mmol), triethylamine (Et₃N, TEA) (34.8 mL, 250 mmol) in anhydrousdichloromethane (DCM) (300 mL) is added at 0° C. (ice bath) in smallportions commercial sulfur trioxide-pyridine complex (Pyr.SO₃) (23.9 g,150 mmol). The reaction mixture is stirred with gradual warming to roomtemperature for about 4-12 hours. The reaction is monitored by TLCand/or LCMS to completion. The majority of volatile is evaporated underreduced pressure and the residue is diluted with 2 M hydrochloric aciduntil acidic. The aqueous phase is extracted with ethyl acetate (EtOAc)(3×). The combined organic extracts are successively washed with asaturated aqueous sodium hydrogencarbonate (NaHCO₃) solution (1×) andwith brine (1×), dried over anhydrous magnesium sulfate (MgSO₄),filtered, and the solvents are evaporated to dryness under reducedpressure. If needed, the crude material is purified by silica gel columnchromatography or is re-crystallized.

Variant B:

Adapting literature known protocol (Aoyama, et al., Synlett, 1998,35-36), commercial activated manganese(IV) oxide (MnO₂) (250-275 mmol)is added at room temperature to a solution of the benzylic alcohol (25mmol) in dichloromethane (DCM) (100 mL). The reaction mixture is stirredfor 12-24 h. The reaction is monitored by TLC and/or LCMS to completion.The reaction mixture is filtered over a short path of Celite® 545 andthe filtrate is concentrated under reduced pressure. The material isoften of sufficient purity to be used directly in the next step withoutfurther isolation and purification. If needed, the crude material ispurified by silica gel column chromatography or is re-crystallized.

Variant C:

Adapting a literature known protocol (Corey and Suggs, TetrahedronLett., 1975, 16(31), 2647-2650; and Fujikawa, et al., J. Am. Chem. Soc.,2008, 130, 14533-14543), to a solution of the benzylic alcohol (20 mmol)in dichloromethane (DCM) (100 mL) is added commercial pyridiniumchlorochromate (Pyr⁺CrO₃Cl⁻, PCC) (28-40 mmol). The reaction mixture isheated to reflux (55° C. oil bath temperature) for 1-4 hours. Thereaction is monitored by TLC and/or LCMS to completion. The reaction iscooled to room temperature. Work-up and product isolation andpurification are conducted as described for Variant B.

Description 3 General Procedure for 3-Amino-3-Arylpropionic Acids ViaRodionov Reaction

Adapting literature known protocols (Tran and Weaver, Tetrahedron, 2002,58, 7449-7461; and Lebedev, et al., Russian J. Gen. Chem, 2005, 75(7),1113-1124), β-amino-3-arylpropionic acids are prepared in one-potaccording to Rodionov by heating a mixture of the aromatic aldehyde (30mmol, malonic acid (30 mmol), and ammonium acetate (NH₄OAc) (4.7 g, 60.7mmol) in ethanol (about 50-100 mL) at reflux for about 12-48 hours (oilbath). The reaction is followed by LC/MS to completion. The reactionmixture is cooled to room temperature upon the target compoundprecipitates generally out. The precipitate is filtered off using aBüchner-funnel and the filter residue is washed with additional EtOH(2×). The collected product is dried under reduced pressure to afford ofthe target compound generally as a colorless solids which are often ofsufficient purity to be used directly in the next step without furtherpurification and isolation procedures.

Description 4 General Procedure for the Preparation of Amino Acid MethylEsters

Adapting literature protocols (Fuchs, et al., U.S. Publication No.2010/144681; and Allison, et al., U.S. Publication No. 2006/069286), thefree (unprotected) or N-(tert-butoxycarbonyl)-protected amino acids (10mmol) is suspended in anhydrous methanol (MeOH) (about 30-80 mL) andcooled to about 0° C. (ice bath). Neat thionyl chloride (SOCl₂) (40-50mmol) is added carefully, and the reaction mixture is heated at refluxfor about 1-6 h before cooling down to room temperature. The reactionwas followed by LC/MS to completion. The solvents are evaporated underreduced pressure using a rotary evaporator. The residue is co-evaporatedwith additional MeOH (2×50) to remove residual volatiles and solvent.Residual solvents are removed under reduced pressure to afford the aminoacid methyl esters generally as colorless solids, which are generally ofsufficient purity to be used directly in the next step without furtherpurification and isolation procedures.

Description 5 General Procedure for the Amino Acid N-Protection withAlkyl Chloroformates

Adapting literature protocols well known in the art, the unprotectedamino acid derivative or a salt thereof, e.g. a hydrochloride salt, (10mmol) is suspended in anhydrous dichloromethane (DCM) (about 30-50 mL)and the mixture is cooled to about 0° C. (ice bath). Neatdiisopropylethylamine (DIPEA, Hünigs-base) (20-50 mmol) is addedfollowed by the appropriate alkyl chloroformate (15 mmol), e.g.,benzylchloroformate (ZCl or CbzCl) or ethylchloroformate, is addeddropwise and the reaction mixture is stirred with gradual warming toroom temperature for overnight. The reaction is monitored by TLC and/orLC/MS to completion. The solvents are removed under reduced pressureusing a rotary evaporator. The residue is diluted with 1.0 molarhydrochloric acid (HCl) and the aqueous phase is extracted with ethylacetate (EtOAc) (3×). The combined organic extracts are dried overanhydrous sodium sulfate (Na₂SO₄) or anhydrous magnesium sulfate(MgSO₄), and filtered using a Büchner funnel. The filter residue iswashed with additional EtOAc, and the combined organic filtrates areevaporated under reduced pressure using a rotary evaporator. The crudematerial is purified by silica gel column chromatography or isre-crystallized to afford the target compounds.

Description 6 General Procedure for the Reduction of Nitro-Aromates toAnilines

Variant A:

Adapting a literature known protocol (Chandrappa, et al., Synlett, 2010,(20), 3019-3022), to a suspension of the nitro aromatic derivative (10mmol) in a mixture of ethanol (EtOH) or methanol (MeOH) with water(10-20 mL alcohol:0.5-3 mL water), iron powder (Fe) (30-100 mmol), andcalcium chloride dihydrate (CaCl₂.2H₂O) (5-10 mmol) are added. Theresulting reaction mixture is heated from about 50° C. to about reflux(oil bath) for about 0.5-3 h. The reaction is followed by TLC (nihydrinstain) and/or analytical LC/MS to completion. The reaction mixture iscooled to room temperature and filtered through a short path of Celite®545 to remove iron residues. The filter aid is washed with additionalalcohol/water mixture or ethyl acetate (EtOAc) (3×). The combinedorganic filtrates are dried over anhydrous sodium sulfate (Na₂SO₄) oranhydrous magnesium sulfate (MgSO₄), the drying agent is filtered off,the filter residue is washed with additional MeOH or EtOAc, filteredover a Büchner funnel, and the combined filtrates are evaporated underreduced pressure using a rotary evaporator. The crude material may bepurified by silica gel column chromatography preferentially usingdichloromethane (DCM) and methanol mixtures optionally containing 1-5vol-% of triethylamine or is re-crystallized.

Variant B:

Adapting literature protocols well known in the art, the nitro aromaticderivative (10 mmol) is dissolved in methanol (MeOH), ethanol (EtOH),ethyl acetate (EtOAc), or mixtures of any of the foregoing (25-50 mL).The heterogeneous catalyst (5 or 10 wt-% palladium on charcoalcontaining ˜50 wt-% water) (about 25-50 wt-% with respect to the nitroaromatic derivative) is added. Optionally, a small amount of acidicadditives, e.g. few drops of HOAc or 1.0 M hydrochloric acid (HCl) areadded to activate the catalyst. The atmosphere is exchanged to hydrogen(3× evacuation/refill technique) and the reaction mixture is stirred atroom temperature under about 15 psi (H₂-balloon) for 1-12 h. Optionally,the reaction is carried out in a stainless steel reactor or aParr-hydrogenation apparatus if higher pressures of H₂ are required. Thereaction is monitored by TLC and/or LCMS to completion. The reactionmixture is filtered over a short plug of Celite® 545, the filtration aidis washed with MeOH, and the combined filtrates are evaporated underreduced pressure. The crude material is purified as described underVariant A.

Description 7 General Procedure for the Reductive N-Alkylation

Adapting literature known protocols (Palani, et al., J. Med. Chem.,2005, 48(15), 4746-4749; van Oeveren, Bioorg. Med. Chem. Lett., 2007,17(6), 1527-1531; Delfourne, et al., Bioorg. Med. Chem., 2004, 12(15),3987-3994; Delfourne, et al., J. Med. Chem., 2002, 47(17), 3765-3771;and Jordan, et al., Bioorg. Med. Chem., 2002, 10(8), 2625-2633), to asolution of the aniline (or a suspension of an aniline addition salt,e.g., a hydrochloride salt) (10 mmol) in methanol (MeOH) (30 mL) atabout 5-15° C. (water bath with some ice) is added trifluoroacetic acid(TFA) (15 mL) (Variant A), acetic acid (15-20 mL) (HOAc) (Variant B), or85 wt-% phosphoric acid (H₃PO₄) (Variant C). To the cooled solution, isadded commercial 2-chloroacetaldehyde (ClCH₂CHO) (˜50 wt-% in water,˜7.87 M) (˜6.5 mL, ˜50 mmol). The reaction mixture is stirred for about15-30 min at this temperature when sodium cyanoborohydride (NaBH₃CN)(2.51 g, 40 mmol) was added in small portions (exothermic hydrogenevolution!). The reaction mixture is stirred for 15-120 min with gradualwarming to room temperature. In some case copious amounts of aprecipitate are generated during the reaction. The reaction is monitoredby TLC and/or LC/MS to completion. The majority of the volatiles(Variants A and B) are evaporated under reduced pressure (rotaryevaporator; ambient to 35° C. bath temperature). The residue isdissolved in ethyl acetate (EtOAc) and the organic phase is successivelywashed with a saturated aqueous solution of sodium hydrogencarbonate(NaHCO₃) (2×) and brine (1×). The organic solution is dried overanhydrous magnesium sulfate (MgSO₄), filtered, and the organic solventswere evaporated to dryness under reduced pressure. If non non-volatileacids are used (Variant C), the reaction mixture is diluted with waterand neutralized (pH 5-7) with solid sodium hydrogencarbonate (NaHCO₃).The aqueous phase is extracted with ethyl acetate (EtOAc) (3×) and thecombined organic extracts are treated as described for Variants A and B.The crude material is purified by silica gel column chromatography or isre-crystallized.

Description 8 General Procedure for Deprotection by Acid Hydrolysis withStrong Aqueous Acids

Adapting literature known protocols (Taylor, et al., Chem. Biol. DrugDes., 2007, 70(3), 216-226; Buss, et al., J. Fluorine Chem., 1986,34(1), 83-114; Abela, et al, J. Chem. Soc., Perkin Trans. 1, 1997, (20),2258-2263; Weisz, et al., Bioorg. Med. Chem. Lett., 1995, 5(24),2985-2988; Zheng, Bioorg., Med., Chem., 2010, 18(2), 880-886; Haines, etal., J. Med. Chem., 1987, 30, 542-547; and Matharu, et al., Bioorg.,Med., Chem., Lett., 2010, 20, 3688-3691), hydrolytic removal ofprotecting groups is conducted through heating a suspension or solutionof the corresponding protected N-mustard (1 mmol) in 2-12 M of anaqueous hydrohalogenic acid (5-10 mL/mmol) or a 20-80 vol-% mixture of a2-12 M of an aqueous hydrohalogenic acid with 1,4-dioxane (5-10 mL/mmol)at an elevated temperature from about 30° C. to about 150° C. (sealedtube) for 1-24 h. The reaction e is be followed by TLC and/or LC/MS tocompletion. Organic side products, e.g., phthalic acid or benzoic acid,may be extracted with an organic solvent, e.g., ethyl acetate (EtOAc) orchloroform (CHCl₃). The aqueous solution or organic volatile solventsare evaporated using a rotary evaporator (40° C. to 60° C. water bathtemperature) to yield the crude target product which may be dissolved ina ˜50 vol-% aqueous acetonitrile (MeCN) followed by lyophilization.Where applicable, the crude target compound is further purified byRP-HPLC purification using acetonitrile/water mixtures containing0.05-0.1 vol-% formic acid (FA) or trifluoroacetic acid (TFA) followedby primary lyophilization, optionally in the presence of 1.0 or anexcess of an acid capable of forming pharmaceutically acceptable saltaddition products. Where applicable, the crude material is purified byre-crystallization, titruation, or repeated precipitation.

Description 9 Global Deprotection of Under Anhydrous Conditions withStrong Acids

Variant A:

Adapting literature known protocols (Springer, et al., J. Med. Chem.,1990, 33(2), 677-681; Davies, et al., J. Med. Chem. 2005, 48(16),5321-5328; Niculesscu-Duvaz, et al., J. Med. Chem., 2004, 47(10),2651-2658; Verny and Nicolas, J. Label. Cmpds, Radiopharm., 1988, 25(9),949-955; Thorn, et al., J. Org. Chem, 1975, 40(11), 1556-1558;Baraldini, et al., J. Med. Chem., 2000, 53(14), 2675-2684; Gourdi, etal., J. Med. Chem., 1990, 33(4), 1177-1186; and Kupczyk-Subotkowska, etal., J. Drug Targeting, 1997, 4(6), 359-370), a solution of thecorresponding protected N,N-bis(2-chloroethyl)aryl-substitutedβ-substituted β-amino acid precursor (1.0 mmol) in neat trifluoroaceticacid (TFA), a mixture of TFA and dichloromethane (DCM) or1,2-dichloroethane (DCE) (90 vol.-% TFA to 90 vol.-% organic solvent),or 98% formic acid (HCO₂H) (10-25 mL/mmol) is stirred at about roomtemperature for about 1-24 h. Optionally, scavengers (2-5 mmol) such astriethysilane (Et₃SiH), triisopropylsilane (iPr₃SiH), thioanisole(PhSMe), or 1,2-dithioethane (HSCH₂CH₂HS) are added to the reactionmixture to suppress unwanted side reactions (Metha, Tetrahedron Lett.,1992, 33(37), 5411-5444). The reaction is be followed by TLC and/oranalytical LC/MS to completion. The solvent is removed under reducedpressure using a rotary evaporator (water bath temperature at about 30°C.). Optionally, residual acid traces are azeotropically removed throughrepeated co-evaporation (5-10×) under reduced pressure using a suitableco-solvent, e.g., ethyl acetate (EtOAc), toluene, or DCM to yield thecrude target compound, which may be used directly in in vitro or in vivoexperiments. Further purification is conducted as described forDescription 8.

Variant B:

Adapting literature known protocols, a solution of the correspondingprotected N,N-bis(2-chloroethyl)aryl-substituted β-substituted γ-aminoacid precursor (1.0 mmol) in 2 M hydrogen chloride in diethyl ether (2.0M HCl in Et₂O) or 4 M hydrogen chloride in 1,4-dioxane (4.0 M HCl in1,4-dioxane) is stirred at about room temperature for about 1-36 h.Optionally scavengers are the same as in Variant A. The reaction is befollowed by TLC and/or analytical LC/MS to completion. The reactionmixture is centrifuged for about 10 min at 3000 rpm, the supernatantdecanted or pipetted off, and the precipitate is suspended in anhydrousEt₂O repeating the centrifugation/washing sequence (2-3×). The crudetarget compound may be used directly in in vitro or in vivo experiments.Further purification is conducted as described for Description 8.

Description 10 General Procedure for the Bromination of BenzylicAlcohols to Benzylic Bromides

Adapting literature known protocols (Harrison and Diehl, Org. Synth.,1955, Coll. Vol. 3, 370), the benzylic alcohol (50 mmol) is dissolved inunhydrous dichloromethane (DCM) (about 100-150 mL) and the solution iscooled to about 0° (ice bath). To the solution is dropwise added acommercial 1.0 M solution of phosphorus tribromide (PBr₃) (50 mmol) andthe resulting mixture is stirred for about 1-2 h at this temperature.The reaction is followed by TLC to completion. The reaction mixture ispoured onto a mixture of crushed ice and a saturated sodiumhydrogencarbonate solution. After phase separation, the aqueous phase isextracted with DCM or ethyl acetate (EtOAc) and the combined organicextracts are washed with a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃) (1×) and brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, the filter residue is washed withDCM, and the combined organic filters are evaporated under reducedpressure. If needed, the crude material is purified by silica gel columnchromatography or is re-crystallized.

Description 11 General Procedure for the Arndt-Eistert Homologation ofAmino Acids

Part A:

Adapting literature protocols (Aldrich Technical Bulletin: Diazald® andDiazomethane Generators; Black, Aldrichchimica Acta, 1983, 16(1), 3-10;and Lombardy, Chem. Ind., 1990, 708), a solution of diazomethane (CH₂N₂)in diethyl ether (Et₂O) is freshly prepared prior to use in an AldrichDiazald® apparatus through addition of a solution of commercialN-methyl-N-nitrosotoluene-4-sulphonamide (Diazald®) (15 g, 70.0 mmol) inEt₂O (150 mL) to a reaction mixture containing potassium hydroxide (KOH)(15 g, 267 mmol) in Et₂O (25 mL), water (30 mL), and2-(2-ethoxyethoxy)ethanol (50 mL) at about 65° C. (oil bath). Thereaction is completed when the yellow color subsided. The CH₂N₂ istrapped in Et₂O.

Part B:

Adapting literature protocols (Podlech and Seebach, Liebigs Ann., 1995,1217-1228; Limbach, et al., Liebigs Ann., 2006, 89(7), 1427-1441;Podlech and Seebach, Angew. Chem. Int. Ed. Engl., 1995, 34(4), 471-472;Müller, et al., Synthesis, 1998, (6), 837-841); and Bartosz-Bechowskiand Konopinska, J. Prakt. Chem., 1989, 331(3), 532-536), an N-protectedamino acid derivative (10 mmol) is dissolved under a nitrogen atmospherein anhydrous tetrahydrofuran (THF) and the solution is cooled to about−20° C. (dry ice/acetone bath). To the solution is addedN-methylmorpholine (NMM) (13 mmol), followed by neat isobutylchloroformate (12 mmol). The reaction mixture is stirred at about −20°C. for about 2 h, when an excess of (5-10 equivalents) of the freshlyprepared ethereal solution of diazomethane is added. Optionally, theprecipitated NMM hydrochloride (NMM-HCl) is filtered off under anitrogen atmosphere prior to diazotation. The reaction mixture isgradually warmed to room temperature and stirred for an additional 2 h.Excess diazomethane is quenched with a few drops of acetic acid (HOAc).The solvents are removed under reduced pressure using a rotaryevaporator. The residue is dissolved in a mixture of Et₂O and ethylacetate (EtOAc). Basic aqueous work-up with a saturated aqueous solutionof sodium hydrogencarbonate (NaHCO₃) and silica gel columnchromatography furnish the diazoketonmes typically as light yellowsolids.

Part C:

Adapting literature protocols (see Part B), an N-protected diazoketone(10 mmol) is dissolved under a nitrogen atmosphere in anhydrous methanol(MeOH) (about 2-4 mL) and anhydrous tetrahydrofuran (THF) (about 20-25mL) and the solution is degassed and placed under a nitrogen atmosphere(3 times evacuation/refill cycling) and under exclusion from (sun)light.A mixture of silver benzoate (AgBz) (5.0 mmol) in THF (about 5-10 mL)and triethylamine (TEA) (20 mmol) is added slowly at room temperature.Gas evolution! The reaction mixture is stirred for about 1-4 hours atroom temperature and concentrated under reduced pressure using a rotaryevaporator. The residue is purified by silica gel column chromatographyusing (EtOAc) and hexane mixtures.

Description 12 General Procedures for the Preparation of SuccinimidylEsters

Adapting a literature protocol (Dexter and Jackson, J. Org. Chem., 1999,64, 7579-7585), to a stirred solution of the N-protected aspartic acidβ-alkyl ester (25 mmol) in ethyl acetate (EtOAc) or acetonitrile (MeCN)(about 25-75 mL) is added solid N-hydroxysuccinimide (NHS, HOSu) (26-28mmol) at about 0° (ice bath). A solution of dicyclohexylcarbodimide(DCC) (25-26 mmol) in EtOAc or MeCN (about 25 mL) is added slowly.Optionally, solid DCC is added in small portions. Optionally, any of thecommon carboxylic acid activation agents can be used for this reaction(Montalbetti and Falque, Tetrahedron, 2005, 61, 10827-10852; and Valeurand M Bradley, Chem. Soc. Rev., 2009, 38, 606-631). The reaction isstirred with gradual warming to room temperature for about 6-24 hours.The reaction is monitored by TLC to completion. The precipitateddicyclohexylurea (DCU) is filtered off using a Büchner-funnel, and thefiltrate is washed with a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃) (3×), brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and evaporated under reducedpressure using a rotary evaporator. The OSu-esters are usually obtainedin quantitative yield and may be of sufficient purity to be useddirectly in the next steps without further isolation and purification.

Description 13 General Procedures for the Reduction of SuccinimidylEsters to Alcohols

Adapting a literature protocol (Dexter and Jackson, J. Org. Chem., 1999,64, 7579-7585), sodium borohydride (NaBH₄) (15-20 mmol) is dissolved inwater (about 3-6 mL) and tetrahydrofuran (about 25-50 mL) at about 0° C.(ice bath). A solution of the succimidyl-ester (10.0 mmol) in THF (about5-10 mL) is added dropwise over about 1 minute. The reaction ismonitored by TLC to completion (<30 min). The reaction is quenchedthrough addition of 1.0 M hydrochloric acid (pH ˜1-2) or a saturatedaqueous solution of ammonium chloride (NH₄Cl). Volatiles (THF) ispartially removed underreduced pressure using a rotary evaporator. Theaqueous phase is extracted with ethyl acetate (EtOAc) (3×). The combinedorganic extracts are washed with a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃) (1×), brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and evaporated under reducedpressure using a rotary evaporator. The residue is purified by silicagel column chromatography using EtOAc and hexane mixtures.

Description 14 General Procedures for the Preparation of Iodides fromAlcohols

Adapting a literature protocol (Dexter and Jackson, J. Org. Chem., 1999,64, 7579-7585), triphenylphosphine (40 mmol), imidazole (40 mmol), andiodine (40 mmol) are added to anhydrous dichloromethane (DCM) (about100-120 mL). A solution of the alcohol (40 mmol) in DCM (about 40 mL) isadded at room temperature. The reaction is monitored by TLC tocompletion (about 1-2 h). The reaction mixture is filtered(Büchner-funnel) to remove precipitated triphenylphosphine oxide (Ph₃PO)and the filtrate is washed with a 1.0 M aqueous solution of sodiumthiosulfate (Na₂S₂O₃) (2×), brine (1×), dried over anhydrous magnesiumsulfate (MgSO₄), filtered, and evaporated under reduced pressure using arotary evaporator. The residue is first slurried in diethyl ether(removal of additional Ph₃PO), filtered through over a short bed ofsilica gel or purified by silica gel column chromatography.

Description 15 General Procedure for the Negishi-Coupling with AromaticHalides

Part A:

Adapting literature protocols (Dexter and Jackson, J. Org. Chem., 1999,64, 7579-7585; Dexter, et al., J. Org. Chem., 2000, 65, 7417-7421;Jackson and M. Perez-Gonzales, Org. Synth., 2005, 81, 77-88; Ross, J.Org. Chem., 2010, 75, 245-248; Anzalone, et al., U.S. Pat. No.8,710,256; Hoepping, et al., International Publication No. WO2014/095739; and Jackson and Perez-Gonzales, Org. Synth., 2005, 81,77-88), zinc dust (Zn) (30 mmol, 3-6 equivalents) is suspended under anatmosphere of inert gas (nitrogen or argon) in anhydrous degassedN,N-dimethylformamide (DMF), N,N-dimethyl acetamide (DMAc or DMA),tetrahydrofuran (THF), or 2-methyl-tetrahydrofuran (2-Me-THF) (about5-10 mL). The zinc metal is activated by addition of elemental iodine(I₂) (about 1.5-3.0 mmol, 15-30 mol-%) and trimethyl silylchloride(MeSiCl, TMSCl) (about 1.5-3.0 mmol, 15-30 mol-%). After subsiding ofthe exotherm, the appropriate iodo-compound (5-10 mmol) is added,optionally as a solution in a small amount of the same anhydrous andegassed solvent, followed by addition of the same amounts of 12 andTMSCl. Optionally, a combination of 1,2-dibromoethane (3 mmol, 30 mol-%)and TMSCl (6 mol %) may be used to activate the zinc dust. Aftersubsiding of the exotherm to room temperature and settling of the zincdust, the supernatend containing the appropriate zinc organic compoundis ready to use in the subsequent Negishi cross-coupling reaction.

Part B:

Adapting literature protocols (see Part A), the supernatand containingthe appropriate zinc organic compound is transferred to a solution ofthe aryl halide (6.5-13 mmol, 1.3 equivalents), tris(benzylideneacetone)dipalladium (Pd₂(dba)₃) (0.125-0.25 mmol, 2.5 mol-%) andtris(o-tolyl)phosphine (P(o-tol)₃) (0.5-1 mmol, 10 mol-%) or SPhos(2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl) (0.25-0.5 mmol, 5mol-%) in anhydrous dry degassed N,N-dimethylformamide (DMF),N,N-dimethyl acetamide (DMAc or DMA), tetrahydrofuran (THF), or2-methyl-tetrahydrofuran (2-Me-THF) (about 5-10 mL). The reactionmixture is stirred at room temperature for 1-12 hours or heated under aninert gas atmosphere to about 40-60° C. for about 1-12 hours. Heating isrequired to cross-couple aryl bromides. He reaction is followed by TLCand/or LCMS to completion. Dilution with water is followed by extractionof the aqueous phase with ethyl acetate (EtOAc) (3×). The combinedorganic extracts are washed with a saturated aqueous solution of sodiumhydrogencarbonate (NaHCO₃) (1×), brine (1×), dried over anhydrousmagnesium sulfate (MgSO₄), filtered, and evaporated under reducedpressure using a rotary evaporator. The residue is purified by silicagel column chromatography using EtOAc and hexane mixtures.

Description 16 General Procedure for the N,N-Bis-(2-Hydroxyethylation)of Anilines with Ethylene Oxide

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al, J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Feau, et al., Org. Biomolecular Chem., 2009, 7(24),5259-5270; Springer, et al., J. Med. Chem., 1990, 33(2), 677-681;Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3), 216-226; Buss, etal., J. Fluorine Chem., 1986, 34(1), 83-114; Larden and Cheung,Tetrahedron Lett., 1996, 37(42), 7581-7582; Spreitzer and Puschmann,Monatshefte für Chemie, 2007, 138(5), 517-522; Niculesscu-Duvaz, et al.,J. Med. Chem., 2004, 47(10), 2651-2658; Weisz, et al., Bioorg. Med.Chem. Lett., 1995, 5(24), 2985-2988; Thorn, et al., J. Org. Chem, 1975,40(11), 1556-1558; Baraldini, et al., J. Med., Chem., 2000, 53(14),2675-2684; Zheng, et al., Bioorg., Med., Chem., 2010, 18(2), 880-886;Gourdi, et al., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al.,J. Med. Chem., 1987, 30, 542-547; Matharu, et al., Bioorg. Med. Chem.Lett., 2010, 20, 3688-3691; and Kupczyk-Subotkowska, et al., J. DrugTargeting, 1997, 4(6), 359-370), a mixture of the corresponding aniline(25.0 mmol) in aqueous acetic acid (HOAc) (25-75 vol-%) (25-100 mL) iscooled to about −20° C. (ice/sodium chloride bath) to about 0° C. (icebath). Optionally, the solvent may also glacial acetic acid (HOAc),water, tetrahydrofuran (THF), ethanol (EtOH), 1,4-dioxane (for highertemperature reactions), or mixtures of any of the foregoing. An excessof ethylene oxide (oxirane) (100-400 mmol) is added to the reactionmixture either neat in pre-cooled form or dissolved in any of theforegoing solvents or mixtures thereof. The reaction mixture is stirredat about room temperature for about 12-48 h. Alternatively, the reactionmixture may be heated in a sealed reaction vessel at 80-140° C. for asimilar time. The reaction is followed by TLC and/or LC/MS and isusually complete when the reaction mixture turns clear. The solvents areremoved under reduced pressure using a rotary evaporator (40-60° C.water bath temperature). The residue is diluted with ethyl acetate(EtOAc), washed with brine, dried over anhydrous magnesium sulfate(MgSO₄) or sodium sulfate (Na₂SO₄), filtered, and the solvents removedunder reduced pressure using a rotary evaporator to yield the targetcompound, which may be used directly in the next step. The crudematerial may be further purified by silica gel column chromatographyusing EtOAc, methanol (MeOH), dichloromethane and hexanes, or mixturesof any of the foregoing to furnish the purified target compound.Alternatively, the crude target compound may be further purified byre-crystallization.

Description 17 General Procedures for Chlorination ofN,N-Bis(2-Hydroxyethyl)-Groups

Variant A: Chlorination with Thionyl Chloride (SOCl₂)

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Jordan, et al., Bioorg. Med. Chem., 2002, 10(8),2625-2633; Abela Medici, et al., J. Chem. Soc., Perkin Trans. 1, 1997,(20), 2258-2263; Taylor, et al., Chem. Biol. Drug Des., 2007, 70(3),216-226; Dheyongera, Bioorg. Med. Chem. 2005, 13(3), 689-698; Zheng,Bioorg. Med. Chem. 2010, 18(2), 880-886; Gourdi, J. Med. Chem., 1990,33(4), 1177-1186; and Lin, et al., Bioorg. Med. Chem. Lett., 2011,21(3), 940-943), to a solution of thionyl chloride (SOCl₂) (10-75 mmol)in an anhydrous organic solvent, e.g., dichloromethane (DCM), chloroform(CHCl₃), 1,2-dichloroethane (DCE), benzene, or mixtures of any of theforegoing (25-100 mL) is added at a temperature from about 0° C. (icebath) to about room temperature the correspondingN,N-bis(2-hydroxyethyl) derivative (5.0 mmol), either in neat form(portions) or as a solution in a small volume in any of the foregoingsolvents. The reaction mixture is stirred at about room temperature toabout 40° C. or heated to reflux for about 10 minutes to about 3 h.Optionally, the reaction is carried out using neat SOCl₂ directly as thesolvent. Optionally, the reaction is carried out in the presence of acatalytic amount of zinc chloride (ZnCl₂) (10 mol-% to 40 mol-%) orN,N-dimethylformamide (about 1 to 3 drops) to facilitate the reaction(Squires, et al., J. Org. Chem., 1975, 40(1), 134-136; and Abela Medici,et al, J. Chem. Soc., Perkin Trans. 1, 1997, (20), 2258-2263). Thereaction is followed by TLC and/or LC/MS to completion. Volatiles(solvents and excess of SOCl₂) are removed under reduced pressure usinga rotary evaporator. Optionally, a small amount of co-solvent, e.g., ofbenzene, is added to assist in azeotropic co-evaporation and removal ofresidual excess chlorination agent. The residue is diluted with 1.0 Mhydrochloric acid (HCl). The aqueous phase is extracted with ethylacetate (EtOAc) (3×), and the combined organic extracts are washed witha saturated aqueous solution of sodium hydrogen carbonate (NaHCO₃) (2×)and brine (1×). The organic layer is dried over anhydrous magnesiumsulfate (MgSO₄) or sodium sulfate (Na₂SO₄), filtered, and the solventsremoved under reduced pressure using a rotary evaporator. The residue ispurified by silica gel column chromatography using EtOAc and hexanesmixtures.

Variant B: Chlorination with Phosphoryl Chloride (POCl₃)

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Feau, et al., Org. Biomolecular Chem., 2009, 7(24),5259-5270; Valu, et al., J. Med. Chem., 1990, 33(11), 3014-3019;Baraldini, et al., J. Med., Chem., 2000, 53(14), 2675-2684; Gourdi, etal., J., Med., Chem., 1990, 33(4), 1177-1186; Haines, et al., J. Med.Chem., 1987, 30, 542-547; and Matharu, et al., Bioorg. Med. Chem. Lett.,2010, 20, 3688-3691), to a solution of phosphorus(V) oxychloride(phosphoryl chloride, POCl₃) (10-50 mmol) in an anhydrous organicsolvent, e.g., benzene, acetonitrile, pyridine, or mixtures of any ofthe foregoing (25-100 mL) is added at a temperature from about 0° C.(ice bath) to about room temperature the correspondingN,N-bis(2-hydroxyethyl) derivative (5.0 mmol) either in neat form(portions) or as a solution in a small volume in any of the foregoingsolvents. The remainder of the reaction, work-up, and product isolationare essentially conducted as described in Variant A.

Variant C: Chlorination with Methanesulfonyl Chloride/Pyridine

Adapting literature known protocols (Jordan, et al., Bioorg. Med. Chem.,2002, 10(8), 2625-2633; Abela Medici, et al, J. Chem. Soc., PerkinTrans. 1, 1997, (20), 2258-2263; Springer, et al., J. Med. Chem., 1990,33(2), 677-681; Larden and Cheung, Tetrahedron Lett., 1996, 37(42),7581-7582), a solution of methanesulfonyl chloride (MsCl) (20.0 mmol) inanhydrous pyridine (about 10 mL) is drop-wise added with stirring and ata temperature of about 0° C. (ice bath) to a solution of thecorresponding N,N-bis(2-hydroxyethyl) derivative (5 mmol) in anhydrouspyridine (about 10 mL). After about 30 minutes, the reaction mixture isheated at 50-100° C. for about 1-3 h. After cooling to room temperature,potential precipitates, if any, e.g., pyridinium methansulfonate, arefiltered off before the solvents are partially removed under reducedpressure using a rotary evaporator. The remainder of the reaction,work-up, and product isolation are essentially conducted as described inVariant A.

Variant D: Chlorination with Triphenylphosphine/Tetrachlorocarbon(PPh₃/CCl₄)

Adapting literature known protocols (Buss, et al., J. Fluorine Chem.,1986, 34(1), 83-114; and Kupczyk-Subotkowska, et al., J. Drug Targeting,1997, 4(6), 359-370), a solution of the correspondingN,N-bis(2-hydroxyethyl) derivative (5 mmol) in anhydrous dichloromethane(DCM) (about 25 mL) containing carbon tetrachloride (CCl₄) (15-25 mmol)is cooled to about 0° C. (ice bath). Alternatively, neat carbontetrachloride (CCl₄) (25 mL) is used as a reaction solvent. The reactionmixture is stirred, and triphenylphosphine (Ph₃P) (10-15 mmol) is addedin portions. The reaction mixture is stirred for about 8-14 h withgradual warming to room temperature. Alternatively, the reaction mixtureis heated at reflux for about 2-6 h. The reaction is followed by TLCand/or LC/MS to completion. The reaction mixture is cooled to roomtemperature and the solvents are removed under reduced pressure using arotary evaporator. The residue is triturated with diethyl ether (Et₂O)(3×) to remove some of the triphenylphosphine oxide (Ph₃PO). The organicphase is evaporated under reduced pressure using a rotary evaporator.The remainder of the reaction, work-up, and product isolation areessentially conducted as described in Variant A.

Description 18 General Procedure for the Mesylation ofN,N-Bis(2-Hydroxyethyl)-Groups

Variant A:

Adapting literature protocols (Davies, et al., J. Med. Chem. 2005,48(16), 5321-5328; Springer, et al., J. Med. Chem., 1990, 33(2),677-681; Niculesscu-Duvaz, et al., J. Med. Chem., 2004, 47(10),2651-2658; and Yang, et al., Tetrahedron, 2007, 63(25), 5470-5476), to acooled solution (about 0° C. (ice bath)) of the correspondingN,N-bis(2-hydroxyethyl) derivative (5.0 mmol) in anhydrousdichloromethane (DCM) (25-50 mL) are added triethylamine (Et₃N, TEA)(25.0 mmol) or anhydrous pyridine (25.0 mmol), and a catalytic amount of4-N,N-(dimethylamino)pyridine (DMAP) (1.0 mmol, 20 mol-%).Methanesulfonyl anhydride (Ms₂O) (20.0 mmol) is added portion-wise or asa solution in DCM (5-10 mL). The reaction mixture is stirred withgradual warming to room temperature for about 8-24 h. The reaction is befollowed by TLC and/or LC/MS. Solvents are removed under reducedpressure using a rotary evaporator. The residue is diluted with 1.0 Mhydrochloric acid (HCl), and the aqueous phase is extracted with ethylacetate (EtOAc) (3×). The combined organic extracts are washed with asaturated aqueous solution of sodium hydrogen carbonate (NaHCO₃), andbrine, dried over anhydrous magnesium sulfate (MgSO₄) or sodium sulfate(Na₂SO₄), filtered, and the solvents are removed under reduced pressureusing a rotary evaporator to yield the target compound, which may beused directly in the next step. Alternatively, the crude residue may befurther purified by silica gel column chromatography using EtOAc,methanol (MeOH), dichloromethane (DCM), and hexanes, or mixtures of anyof the foregoing to furnish the purified target compound. Alternatively,the crude target compound may be further purified by re-crystallization.

Variant B:

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; B. D. Palmer, et al., J. Med. Chem., 1994, 37,2175-2184; Palmer, et al., J. Med. Chem, 1996, 39(13), 2518-2528;Spreitzer and Puschmann, Monatshefte für Chemie, 2007, 138(5), 517-522;Lin, et al., Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943; Gourdi, etal., J. Med. Chem., 1990, 33(4), 1177-1186; Ferlin, et al., Bioorg. Med.Chem., 2004, 12(4), 771-777; Thorn, et al., J. Org. Chem, 1975, 40(11),1556-1558; Coggiola, et al., Bioorg. Med. Chem. Lett., 2005, 15(15),3551-3554), to a cooled solution (about 0° C. (ice bath)) of thecorresponding N,N-bis(2-hydroxyethyl) derivative (5.0 mmol) in anhydrousdichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate (EtOAc), ora mixture thereof (20-40 mL) are added triethylamine (Et₃N, TEA) (15.0mmol) or anhydrous pyridine (25.0 mmol). Methanesulfonyl chloride (MsCl)(12.5 mmol) is added drop-wise to the reaction mixture. The reactionmixture is stirred for about 1-2 h at this temperature. The reaction maybe followed by TLC and/or LC/MS. Aqueous work-up and purification bysilica gel chromatography are performed as described for Variant A.

Description 19 General Procedure for the Finkelstein Conversion toN,N-Bis(2-Halogenoethyl)-Groups

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Palmer, et al., J. Med. Chem., 1994, 37, 2175-2184;Palmer, et al., J. Med. Chem., 1996, 39(13), 2518-2528; Davies, et al.,J. Med. Chem. 2005, 48(16), 5321-5328; Niculesscu-Duvaz, et al., J. Med.Chem., 2004, 47(10), 2651-2658; Weisz, et al., Bioorg. Med. Chem. Lett.,1995, 5(24), 2985-2988; Thorn, J. Org. Chem, 1975, 40(11), 1556-1558;Lin, et al., Bioorg. Med. Chem. Lett., 2011, 21(3), 940-943; Gourdi, etal., J. Med. Chem. 1990, 33(4), 1177-1186; Yang, et al., Tetrahedron,2007, 63(25), 5470-5476; Ferlin, et al., Bioorg. Med. Chem., 2004,12(4), 771-777; and Coggiola, et al., Bioorg. Med. Chem. Lett., 2005,15(15), 3551-3554), a slurry of the correspondingN,N-bis(2-methylsulfonyloxyethyl) derivative (5.0 mmol) and an alkalimetal halide, e.g., lithium chloride (LiCl), lithium bromide (LiBr),sodium chloride (NaCl), sodium bromide (NaBr), or sodium iodide (NaI)(20-80 mmol) in an anhydrous organic solvent, e.g.,N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), acetone,2-butanone (methyl ethyl ketone, MEK), 3-methyl-2-butanone (isopropylmethyl ketone, MIPK), acetonitrile (MeCN), methanol (MeOH),tetrahydrofuran (THF), ethyl acetate (EtOAc) or a mixture of any of theforegoing (10-30 mL), is stirred at room temperature or heated at50-150° C. for about 1-12 h. The reaction is followed by TLC and/orLC/MS to completion. Solvents are partially or completely removed underreduced pressure using a rotary evaporator. The residue is diluted with1.0 M hydrochloric acid (HCl), and the aqueous phase is extracted withethyl acetate (EtOAc) (3×). The combined organic extracts are washedwith a saturated aqueous solution of sodium hydrogen carbonate (NaHCO₃),and brine, dried over anhydrous magnesium sulfate (MgSO₄) or sodiumsulfate (Na₂SO₄), filtered, and the solvents are removed under reducedpressure using a rotary evaporator to yield the target compound, whichmay be used directly in the next step. Alternatively, the crude residuemay be further purified by silica gel column chromatography using EtOAc,methanol (MeOH), dichloromethane (DCM), and hexanes, or mixtures of anyof the foregoing to furnish the purified target compound. Alternatively,the crude target compound may be further purified by re-crystallization.

Example 13-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoic Acid(1)

Step A: (2-Methyl-5-nitro-phenyl)methanol (1a)

Following the General Procedure of Description 1,2-methyl-5-nitro-phenyl)methanol (1a) was prepared from commercial2-methyl-5-nitro benzoic acid (50.0 g, 276 mmol) with boranedimethylsulfide complex (2.0 M BH₃.SMe₂ in THF) (166 mL, 332 mmol) inanhydrous tetrahydrofuran (400 mL) to yield 44.0 g (˜quantitative yield)of the target compound (1a) as a pale yellow solid which was ofsufficient purity to be used directly in the next step without furtherisolation and purification. R_(f): ˜0.50 (EtOAc/Hxn=1:1 v/v). ¹H NMR(300 MHz, CDCl₃): δ 8.30 (d, J=2.4 Hz, 1H), 8.05 (dd, J=8.4, 2.4 Hz,1H), 7.31 (d, J=8.1 Hz, 1H), 4.78 (d, J=5.1 Hz, 2H), 2.41 (s, 3H), 1.87(br. t, J=5.1 Hz, 1H) ppm. The compound is also commercially available.

Step B: 2-Methyl-5-nitro-benzaldehyde (1b)

Following the General Procedure of Description 2 (Variant A),2-methyl-5-nitro-benzaldehyde (1b) (Beech, J. Chem. Soc. (C), 1967,2374-2375) was prepared from 2-methyl-5-nitro-phenyl)methanol (1a) (16.3g, 97.3 mmol) in the presence of dimethylsulfoxide (DMSO) (56.8 mL, 62.6g, 0.80 mol), triethylamine (TEA, Et₃N) (69.5 mL, 50.6 g, 0.50 mmol),and sulfur trioxide pyridine complex (SO₃.pyridine) (47.8 g, 0.30 mol)in dichloromethane (600 mL). Purification by silica gel columnchromatography using a mixture of ethyl acetate (EtOAc) and hexane(EtOAc/hexane=1:4 v/v) afforded 12.6 g (78% yield) of the targetcompound (1b) as a yellow-beige solid.

Following the General Procedure of Description 2 (Variant B),2-methyl-5-nitro-benzaldehyde (1b) (Beech, J. Chem. Soc. (C), 1967,2374-2375) was prepared from 2-methyl-5-nitro-phenyl)methanol (1b) (4.03g, 24.1 mmol) in the presence of manganese dioxide (MnO₂) (22 g, 254mmol) in dichloromethane (DCM) (100 mL). Work-up afforded 3.56 g (89%yield) of the target compound (1b) as a pale yellow to beige solid. Thematerial was of sufficient purity to be used directly in the next stepwithout further isolation and purification.

Following the General Procedure of Description 2 (Variant C),2-methyl-5-nitro-benzaldehyde (1b) (Beech, J. Chem. Soc. (C), 1967,2374-2375) was prepared from 2-methyl-5-nitro-phenyl)methanol (1a) (5.00g, 29.9 mmol) in the presence of pyridinium chlorochromate (PCC) (9.02g, 41.9 mmol) in dichloromethane (DCM) (150 mL). Purification by silicagel column chromatography using mixtures of ethyl acetate (EtOAc) andhexane (EtOAc/hexane=1:4 v/v→EtOAc/hexane=1:4 v/v) afforded 4.67 g (94%yield) of the target compound (1b) as a yellow-beige solid. R_(f): ˜0.76(EtOAc/Hxn=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 10.32 (s, 1H), 8.65 (dd,J=2.7 Hz, 1H), 8.31 (dd, J=8.4, 2.4 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H),2.79 (s, 3H) ppm. The compound is also commercially available.

Step C: 3-Amino-3-(2-methyl-5-nitro-phenyl)propanoic Acid (1c)

Following the General Procedure of Description 3,3-amino-3-(2-methyl-5-nitro-phenyl)propanoic acid (1c) was prepared from2-methyl-5-nitro-benzaldehyde (1b) (5.0 g, 30.3 mmol), malonic acid (3.2g, 30.3 mmol), and ammonium acetate (NH₄OAc) (4.7 g, 60.7 mmol) inethanol (EtOH) (70 mL) at reflux for 48 hours (oil bath). The reactionwas followed by LC/MS to completion. Filtrative work-up afforded 2.2 g(32% yield) of the target compound (1c) as a colorless solid which wasof sufficient purity to be used directly in the next step withoutfurther purification and isolation procedures. ¹H NMR (300 MHz, D₂O): δ8.20 (d, J=2.4 Hz, 1H), 8.01 (dd, J=8.1, 2.1 Hz, 1H), 7.38 (d, J=8.7 Hz,1H), 4.84 (t, J=6.9 Hz, 1H), 2.80-2.60 (m, 2H), 2.37 (s, 3H) ppm. LC/MS:R_(t)=0.480 min; ESI (pos.) m/z=225.1 (M+H⁺)⁺, ESI (neg.) m/z=223.0(M−H⁺)⁻, 447.1 (2M−H⁺)⁻.

Step D: Methyl β-amino-3-(2-methyl-4-nitro-phenyl)propanoateHydrochloride (1d)

Following the General Procedure of Description 4, methylβ-amino-3-(2-methyl-4-nitro-phenyl)propanoate hydrochloride (1d) wasprepared in a suspension in anhydrous methanol (MeOH) (40 mL) fromβ-amino-3-(2-methyl-5-nitro-phenyl)propanoic acid (1c) (2.2 g, 9.81mmol) with neat thionyl chloride (SOCl₂) (3.54 mL, 5.8 g, 49.1 mmol).Evaporative work-up afforded 2.73 g (about quantitative yield) of thetarget compound (1d) as a colorless solid, which was of sufficientpurity to be used directly in the next step without further purificationand isolation procedures. ¹H NMR (300 MHz, DMSO-d⁶): δ 8.86 (br. s, 3H),8.60 (d, J=2.1 Hz, 1H), 8.11 (dd, J=8.4, 2.1 Hz, 1H), 7.53 (d, J=8.4 Hz,1H), 4.86 (br. m, 1H), 3.53 (s, 3H), 3.29 (dd, J=16.8, 6.0 Hz, 1H), 3.13(dd, J=16.8, 8.7 Hz, 1H) ppm. LC/MS: R_(t)=0.492 min; ESI (pos.)m/z=239.1 (M+H⁺)⁺.

Step E: Methyl3-benzyloxycarbonylamino-3-(2-methyl-5-nitro-phenyl)propanoate (1e)

Following the General Procedure of Description 5, methyl3-benzyloxycarbonylamino-3-(2-methyl-5-nitro-phenyl)propanoate (1e) wasprepared from crude methyl β-amino-3-(2-methyl-4-nitro-phenyl)propanoatehydrochloride (1d) (2.7 g, 9.81 mmol), benzyl chloroformate (ZCl, CbzCl)(2.20 mL, 2.63 g of 95% purity=2.5 g, 14.7 mmol), anddiisopropylethylamine (DIPEA, Hünigs-base) (6.87 mL, 5.1 g, 39.2 mmol)in anhydrous dichloromethane (DCM) (50 mL). Acidic aqueous work-up andpurification by silica gel column chromatography afforded 3.4 g (92%yield) of the target compound (1e) as a colorless solid. R_(f)=0.44(EtOAc/Hxn=1:2 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.16 (d, J=2.7 Hz, 1H),8.24 (dd, J=8.4, 2.4 Hz, 1H), 7.38-7.26 (m, 6H), 5.86 (br. d, 1H),5.42-5.36 (br. m, 1H), 5.09 (d, J=12.0 Hz, 1H), 5.04 (d, J=12.0 Hz, 1H),3.64 (s, 3H), 2.84-2.78 (br. m, 2H) ppm. LC/MS: R_(t)=1.790 min; ESI(pos.) m/z=373.2 (M+H⁺)⁺, 767.6 (2M+Na⁺)⁺, ESI (neg.) m/z=743.2(2M−H⁺)⁻.

Step F: Methyl3-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-propanoate (1f)

Following the General Procedure for of Description 6 (Variant A), methyl3-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-propanoate (1f) wasprepared from methyl3-benzyloxycarbonylamino-3-(2-methyl-5-nitro-phenyl)propanoate (1e)(3.35 g, 8.99 mmol), iron powder (Fe) (4.5 g, 81.1 mmol), and calciumchloride dihydrate (CaCl₂.2H₂O) (0.6 g, 4.05 mmol) in a mixture ofmethanol (MeOH)/water (68 mL: 12 mL v/v). The reaction mixture washeated at reflux for 2 hours (oil bath). Removal of the iron residues byfiltration and compound isolation procedures yielded 3.1 g (aboutquantitative yield) of the target compound (1f) as a light yellow solidwhich was of sufficient purity to be used directly in the nest stepwithout further isolation and purification. ¹H NMR (300 MHz, DMSO-d⁶): δ7.85 (d, J=8.1 Hz, 1H), 7.36-7.24 (m, 5H), 6.74 (d, J=7.8 Hz, 1H), 6.51(d, J=2.1 Hz, 1H), 6.33 (dd, J=8.4, 2.4 Hz, 1H), 5.10-5.00 (m, 1H), 4.98(d, J=12.3 Hz, 1H), 4.92 (d, J=12.9 Hz, 1H), 4.79 (br. s, 2H), 3.54 (s,3H) ppm. LC/MS: R_(t)=1.072 min; ESI (pos.) m/z=365.1 (M+Na⁺)⁺, 685.2(2M+Na⁺)⁺, 702.2 (2M+Na⁺)⁺.

Step G: Methyl3-benzyloxycarbonylamino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoate(1g)

Following the General Procedure for of Description 7 (Variant A), methyl3-benzyloxycarbonylamino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoate(1g) was prepared from methyl3-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-propanoate (1f)(3.1 g, 9.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M)(5.8 mL, 45.6 mmol), and sodium cyanoborohydride (NaBH₃CN) (2.4 g of 95%purity=2.3 g, 36.6 mmol) in a mixture of methanol (MeOH) (60 mL) andtrifluoroacetic acid (TFA) (30 mL). Aqueous work-up and purification bysilica gel column chromatography with an ethyl acetate (EtOAc) hexanemixture (EtOAc/hexane=1:2, v/v) afforded 2.90 g (69% yield) of the titlecompound (1g) as a colorless solid. R_(f)=0.55 (EtOAc/hexane=1:2, v/v,ninhydrine negative). ¹H NMR (300 MHz, CDCl₃): δ 7.40-7.32, (br. m, 5H),7.03 (d, J=8.4 Hz, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.52 (dd, J=8.4, 2.7 Hz,1H), 5.78-5.62 (br. m, 1H), 5.34-5.26 (m, 1H), 5.09 (d, J=12.6 Hz, 1H),5.07 (d, J=12.6 Hz, 1H), 3.78-3.54 (m, 11H), 2.84-2.78 (m, 2H) ppm.LC/MS: R_(t)=2.271 min; ESI (pos.) m/z=467.1 (M+H⁺)⁺, 489.1 (M+Na⁺)⁺.LC/UV: R_(t)=12.939 min, 100.0% AUC at λ=254 nm.

Step H: 3-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoicAcid (1)

Following the General Procedure of Description 8,3-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoic acid(1) was prepared through hydrolytic deprotection of methyl3-benzyloxycarbonylamino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoate(1g) (2.9 g, 6.2 mmol) in a mixture of concentrated hydrochloric acid(HCl) (20 mL) and 1,4-dioxane (20 mL) at about 100° C. (oil bath) in 48hours. The residue was purified by preparative HPLC, immediately frozenafter collection, followed by primary lyophilization to afford 728 mg(33% yield) of the target compound (1) as a colorless solid. ¹H NMR (300MHz, DMSO-d⁶): δ 6.98 (d, J=8.4 Hz, 1H), 6.85 (d, J=2.4 Hz, 1H), 6.56(dd, J=8.4, 2.4 Hz, 1H), 4.36 (dd, J=9.9, 4.5 Hz, 1H), 3.56-3.53 (br. m,8H), 2.48-2.44 (m, 2H) ppm. LC/MS: R_(t)=1.226 min; ESI (pos.) m/z=319.2(M+H⁺)⁺, ESI (neg.) m/z=316.9 (M−H⁺)⁻, 635.1 (2M−H⁺)⁻. LC/UV:R_(t)=6.723 min, 99.3% AUC at λ=254 nm. Various batches of mono- ordihydrochloride salts of (1) were prepared by primary lyophilization ofsolutions of (5) in aqueous acetonitrile (MeCN) containing either 1.0eq. of 1.0 N hydrochloric acid (HCl) or an excess of 1.0 N or higherconcentrated hydrochloric acid (HCl).

Example 23-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoic Acid(2)

Step A: (2-Methyl-4-nitro-phenyl)methanol (2a)

Following the General Procedure of 1, 2-methyl-4-nitro-phenyl)methanol(2a) was prepared from commercial 2-methyl-4-nitro benzoic acid (5.0 g,27.6 mmol) with borane dimethylsulfide complex (2.0 M BH₃.SMe₂ in THF)(27.6 mL, 55.2 mmol) in anhydrous tetrahydrofuran (100 mL) to yield 4.62g (quantitative yield) of the target compound (7a) as a pale yellowsolid which was of sufficient purity to be used directly in the nextstep without further isolation and purification. R_(f): ˜0.50(EtOAc/Hxn=1:1 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.07 (dd, J=8.4, 2.1 Hz,1H), 8.02 (d, J=2.1 Hz, 1H), 7.62 (d, J=8.1 Hz, 1H), 4.79 (s, 2H), 2.38(s, 3H), 1.87 (br. s, 1H) ppm. The spectroscopic data correspond to thedata provided in the literature. The compound is also commerciallyavailable.

Step B: 2-Methyl-4-nitro-benzaldehyde (2b)

Following the General Procedure of Description 2 (Variant B),2-methyl-4-nitro-benzaldehyde (2b) was prepared from2-methyl-4-nitro-phenyl)methanol (1a) (8.4 g, 50.3 mmol) in the presenceof manganese dioxide (MnO₂) (48.1 g, 553 mmol). Work-up afforded 7.5 g(90% yield) of the target compound (7b) as a yellow solid. The materialwas of sufficient purity to be used directly in the next step withoutfurther isolation and purification. R_(f): ˜0.58 (EtOAc/Hxn=1:2 v/v). ¹HNMR (300 MHz, CDCl₃): δ 10.39 (s, 1H), 8.20 (dd, J=8.4, 2.1 Hz, 1H),8.14 (br. s, 1H), 7.98 (d, J=8.1 Hz, 1H), 2.79 (s, 3H) ppm. Thespectroscopic data correspond to the data provided in the literature.The compound is also commercially available.

Step C: 3-Amino-3-(2-methyl-4-nitro-phenyl)propanoic Acid (2c)

Following the General Procedure of Description 3,3-amino-3-(2-methyl-4-nitro-phenyl)propanoic acid (2c) was prepared from2-methyl-4-nitro-benzaldehyde (2b) (800 mg, 5.0 mmol), malonic acid (520mg, 5.0 mmol), and ammonium acetate (NH₄OAc) (578 mg, 7.5 mmol) inethanol (EtOH) (10 mL) at reflux for 48 h (oil bath). The reaction wasfollowed by LC/MS to completion. Filtrative work-up afforded 510 mg (45%yield) of the target compound (1c) as a near colorless solid which wasof sufficient purity to be used directly in the next step withoutfurther purification and isolation. ¹H NMR (300 MHz, D₂O): δ 8.01-7.97(m, 2H), 7.46 (d, J=8.4 Hz, 1H), 4.83 (t, J=7.2 Hz, 1H), 2.70-2.65 (m,2H), 2.33 (s, 3H) ppm. LC/MS: R_(t)=1.274 min; ESI (pos.) m/z=225.1(M+H⁺)⁺.

Step D: Methyl β-amino-3-(2-methyl-4-nitro-phenyl)propanoateHydrochloride (2d)

Following the General Procedure of Description 4, methylβ-amino-3-(2-methyl-4-nitro-phenyl)propanoate hydrochloride (2d) wasprepared in a suspension in anhydrous methanol (MeOH) (10 mL) fromβ-amino-3-(2-methyl-4-nitro-phenyl)propanoic acid (2c) (510 mg, 2.27mmol) with neat thionyl chloride (SOCl₂) (2.0 mL, 3.28 g, 27.5 mmol).Evaporative work-up afforded 2.73 g (about quantitative yield) of thetarget compound (1d) as a colorless solid, which was of sufficientpurity to be used directly in the next step without further purificationand isolation. LC/MS: R_(t)=0.508 min; ESI (pos.) m/z=239.1 (M+H⁺)⁺.

Step E: Methyl3-(ethoxycarbonylamino)-3-(2-methyl-4-nitro-phenyl)propanoate (2e)

Following the General Procedure of Description 5, methyl3-(ethoxycarbonylamino)-3-(2-methyl-4-nitro-phenyl)propanoate (2e) wasprepared from crude methyl β-amino-3-(2-methyl-4-nitro-phenyl)propanoatehydrochloride (2e) (624 mg, 2.27 mmol), ethyl chloroformate (EtOCOCl)(327 μL, 371 mg 3.42 mmol), and diisopropylethylamine (DIPEA,Hünigs-base) (1.12 mL, 885 mg, 6.84 mmol) in anhydrous dichloromethane(DCM) (10 mL). Silica gel column chromatography afforded 701 mg (aboutquantitative yield) of the target compound (2e) as a colorless solid.R_(f)=0.42 (EtOAc/Hxn=1:1 v/v).

Step F: Methyl3-(4-amino-2-methyl-phenyl)-3-(ethoxycarbonylamino)propanoate (2f)

Following the General Procedure of Description 6 (Variant B), methyl3-(4-amino-2-methyl-phenyl)-3-(ethoxycarbonylamino)propanoate (2f) isprepared from methyl3-(ethoxycarbonylamino)-3-(2-methyl-4-nitro-phenyl)propanoate (2e) (701mg, 2.26 mmol) through hydrogenation (about 15 psi; H₂-filled balloon)in the presence 10 wt-% Pd/C containing 50-wt-% water (˜70 mg) and atroom temperature for about 12 hours to afford 632 mg (about quantitativeyield) of the target compound (2f) as a brownish oil, which was ofsufficient purity to be used in the next step without additionalpurification and isolation. LC/MS: R_(t)=0.533 min; ESI (pos.) m/z=303.1(M+H⁺)⁺.

Step G: Methyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(ethoxycarbonylamino)-propanoate(2g)

Following the General Procedure for of Description 7 (Variant A), methyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(ethoxycarbonylamino)-propanoate(2g) was prepared from methyl3-(4-amino-2-methyl-phenyl)-3-(ethoxycarbonylamino)propanoate (2f) (632mg, 2.26 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (1.44mL, 11.3 mmol), and sodium cyanoborohydride (NaBH₃CN) (598 mg of 95%purity=568 g, 9.04 mmol) in a mixture of methanol (MeOH) (20 mL) andtrifluoroacetic acid (TFA) (10 mL). Purification by silica gel columnchromatography with an ethyl acetate (EtOAc)/hexane mixture(EtOAc/hexane=1:1, v/v) afforded 714 mg (78% yield) of the titlecompound (2g) as a colorless solid. R_(f)=0.54 (EtOAc/Hxn=1:2 v/v,ninhydrine negative). ¹H NMR (300 MHz, CDCl₃): δ 7.11 (d, J=8.4 Hz, 1H),6.49 (dd, J=8.7, 2.7 Hz, 1H), 6.44 (d, J=2.4 Hz, 1H), 5.36-5.22 (m, 2H),4.08 (q, J=7.2 Hz, 2H), 3.76-3.54 (m, 11H), 2.90-2.70 (m, 2H), 2.39 (s,3H), 1.21 (t, J=7.2 Hz, 3H) ppm. LC/MS: R_(t)=2.174 min; ESI (pos.)m/z=405.1 (M+H⁺)⁺.

Step H: 3-Amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoicAcid (2)

Following the General Procedure of Description 8,3-amino-3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propanoic acid(2) was prepared through hydrolytic deprotection of methyl3-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(ethoxycarbonylamino)-propanoate(2g) (150 mg, 0.37 mmol) in concentrated hydrochloric acid (HCl) (5 mL)at about 100° C. (oil bath) in 48 h. The residue was partially purifiedby preparative HPLC, immediately frozen after collection, followed byprimary lyophilization to afford 40 mg of the target compound (1) as acolorless solid. ¹H NMR (300 MHz, DMSO-d⁶): δ 7.30 (d, J=6.3 Hz, 1H),6.63 (dd, J=6.6, 2.1 Hz, 1H), 6.56 (d, J=1.8 Hz, 1H), 4.55 (t, J=5.7 Hz,1H), 3.76-3.62 (br. m, 8H), 2.84 (dd, J=12.3, 5.1 Hz, 1H), 2.71 (dd,J=12.0, 5.7 Hz, 1H), 2.29 (s, 3H) ppm. LC/MS: R_(t)=1.094 min; ESI(neg.) m/z=317.0 (M−H⁺)⁻. LC/UV: R_(t)=7.393 min, 98.6% AUC at λ=254 nm.

Example 33-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic Acid (3)

Step A: 2-(Bromomethyl)-1-methyl-4-nitro-benzene (3a)

Following the General Procedure of Description 10,2-(bromomethyl)-1-methyl-4-nitro-benzene (3a) was prepared throughbromination of (2-methyl-5-nitro-phenyl)methanol (1a) (11.0 g, 65.8mmol) (prepared as described in Example 1) dissolved in dichloromethane(DCM) (110 mL) with a solution of phosphorus tribromide (PBr₃) in (1.0 MPBr₃ in DCM) (65.8 mL). Aqueous work-up yielded 11.3 g (75% yield) of alight yellow solid which was of sufficient purity to be used directlyand without further isolation and purification in the next step.R_(f)=0.56 (EtOAc/Hxn=1:5 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.19 (d,J=2.4 Hz, 1H), 8.07 (dd, J=8.4, 2.7 Hz, 1H), 7.36 (d, J=8.7 Hz, 1H),4.53 (s, 2H), 2.52 (s, 2H) ppm. The spectroscopic data correspond to thedata provided in the literature. The compound is also commerciallyavailable.

Step B: Diethyl2-acetamido-2-[(2-methyl-5-nitro-phenyl)methyl]propanedioate (3b)

Adapting a literature protocol (Haudegond, et al., J. Org. Chem., 1979,44(17), 3063-3065), an ethanolic solution of sodium ethanolate (NaOEt)(35.6 mmol) was freshly prepared from elemental sodium (Na) (819 mg,35.6 mmol) in anhydrous ethanol (EtOH) (80 mL) under an atmosphere ofnitrogen at room temperature. When the H₂-evolution was ceased,commercial diethyl 2-acetamidopropanedioate (7.9 g, 36.4 mmol) was addedin small portions. The reaction mixture was heated at about 75° C. (oilbath) for about 30 min before 2-(bromomethyl)-1-methyl-4-nitro-benzene(3a) (8.2 g, 35.6 mmol) was added, and the reaction mixture was heatedat reflux (oil bath) for about 10 h. The reaction was followed by LC/MSto completion. The solid was collected by filtration using aBüchner-funnel and the residue was washed successively with EtOH (2×)and ethyl acetate (EtOAc) (1×), and dried under reduced pressure toafford 8.4 g (64% yield) of the target compound diethyl2-acetamido-2-[(2-methyl-5-nitro-phenyl)methyl]propanedioate (3b) as acolorless solid. ¹H NMR (300 MHz, DMSO-d⁶): δ 8.29 (s, 1H), 8.00 (dd,J=8.1, 2.4 Hz, 1H), 7.72 (d, J=2.4 Hz, 1H), 7.45 (d, J=8.7 Hz, 1H), 4.15(q, J=7.2 Hz, 4H), 3.58 (s, 2H), 2.26 (s, 3H), 1.90 (s, 3H), 1.17 (t,J=7.2 Hz, 6H) ppm. LC/MS: R_(t)=1.818 min; ESI (pos.) m/z=367.1 (M+H⁺)⁺,755.3 (2M+Na⁺)⁺.

Step C: 2-Amino-3-(2-methyl-5-nitro-phenyl)propanoic Acid Hydrochloride(3c)

Following the General Procedure of Description 8,2-amino-3-(2-methyl-5-nitro-phenyl)propanoic acid hydrochloride (3c) wasprepared by acid hydrolysis of diethyl2-acetamido-2-[(2-methyl-5-nitro-phenyl)methyl]propanedioate (3b) (8.4g, 22.9 mmol) with concentrated (˜37 wt-%) hydrochloric acid (HCl) (150mL). The suspension was heated at reflux (oil bath) for about 6 h. Thereaction was followed by LC/MS to completion. The cooled clear solutionwas evaporated under reduced pressure using a rotary evaporator to yield6.7 g (about quantitative yield) of the target compound (3c) as acolorless solid. ¹H NMR (300 MHz, DMSO-d⁶): δ 8.58 (br. s, 3H), 8.12 (d,J=2.1 Hz, 1H), 8.03 (dd, J=8.4, 2.4 Hz, 1H), 7.47 (d, J=8.7 Hz, 1H),4.20-4.10 (m, 1H), 3.25 (d, J=7.2 Hz, 2H), 2.42 (s, 3H) ppm. LC/MS:R_(t)=0.705 min; ESI (pos.) m/z=225.1 (M+H⁺)⁺, 449.1 (2M+H⁺)⁺; ESI(neg.) m/z=223.0 (M−H⁺)⁻, 447.1 (2M−H⁺)⁻.

Step D: 2-Benzyloxycarbonylamino-3-(2-methyl-5-nitro-phenyl)propanoicAcid (3d)

Adapting a literature protocol,2-benzyloxycarbonylamino-3-(2-methyl-5-nitro-phenyl)propanoic acid (3d)was prepared from 2-amino-3-(2-methyl-5-nitro-phenyl)propanoic acidhydrochloride (3c) (6.7 g, 25.7 mmol) in 1,4-dioxane (50 mL) and a 10wt-% aq. solution of sodium hydroxide (NaOH) (˜3.75 M, 13.7 mL, 51.4mmol) at about 0° C. (ice bath). Water (32 mL) was added followed bysolid sodium hydrogencarbonate (NaHCO₃) (2.15 g, 25.7 mmol), andcommercial benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (CbzOSu) (6.4 g,25.7 mmol). The reaction mixture was stirred overnight at roomtemperature. The volatiles were removed under reduced pressure using arotary evaporator. Acid work up at a pH of about 3 and tritruation ofthe crude product with ethyl acetate (EtOAc) and hexane (Hxn)(EtOAc/Hxn=3:7) at about 50° C. (oil bath), the solid was collected byfiltration (Büchner-funnel) to afford 6.1 g (65% yield) of the targetcompound (3d) as a colorless solid. ¹H NMR (300 MHz, CDCl₃): δ 8.02-7.98(m, 2H), 7.40-7.21 (m, 6H), 5.33 (d, J=8.4 Hz, 1H), 5.06 (d, J=12.0 Hz,1H), 5.03 (d, J=12.0 Hz, 1H), 4.74-4.70 (m, 1H), 3.57 (dd, J=14.7, 5.4Hz, 1H), 3.08 (dd, J=14.4, 7.8 Hz, 1H), 2.45 (s, 3H) ppm. LC/MS:R_(t)=1.812 min; ESI (neg.) m/z=357.1 (M−H⁺)⁻, 715.1 (2M−H⁺)⁻.

Step E: BenzylN-[3-diazo-1-[(2-methyl-5-nitro-phenyl)methyl]-2-oxo-propyl]carbamate(3e)

Following the general procedure of Description 11 (Part A), a solutionof diazomethane (CH₂N₂) in diethyl ether (Et₂O) was freshly preparedprior to use in an Aldrich Diazald® apparatus from commercialN-methyl-N-nitrosotoluene-4-sulphonamide (Diazald®) (15 g, 70.0 mmol),potassium hydroxide (KOH) (15 g, 267 mmol) in a mixture of Et₂O (25 mL),water (30 mL), and 2-(2-ethoxyethoxy)ethanol (50 mL) at about 65° C.(oil bath). The etheral distillate was trapped in Et₂O (150 mL) in Et₂O(150 mL).

Following the general procedure of Description 11 (Part B), the mixedanhydride of (3d) is prepared from2-benzyloxycarbonylamino-3-(2-methyl-5-nitro-phenyl)propanoic acid (3d)(3.0 g, 8.38 mmol), N-methylmorpholine (NMM) (1.20 mL, 1.1 g, 10.9mmol), neat isobutyl chloroformate (1.34 mL, 1.4 g, 10.1 mmol) at about−20° C. (dry ice/acetone bath) under a nitrogen atmosphere. After the 2hours −20° C., an excess of (˜6 equivalents) of the freshly preparedethereal solution of diazomethane was added (˜100 mL). Aqueous work andpurification by silica gel column chromatography (EtOAc/Hxn=2:3 v/v)afforded 2.5 g (85% yield of the target compound benzylN-[3-diazo-1-[(2-methyl-5-nitro-phenyl)methyl]-2-oxo-propyl]carbamate(3e) as a light yellow solid. R_(f)=0.25 (EtOAc/Hxn=2:3 v/v). ¹H NMR(300 MHz, CDCl₃): δ 8.02-7.98 (m, 2H), 7.40-7.24 (m, 6H), 5.46 (d, J=8.4Hz, 1H), 5.29 (s, 1H), 5.05 (d, J=12.0 Hz, 1H), 5.02 (d, J=12.6 Hz, 1H),4.52-4.46 (m, 1H), 3.23 (dd, J=14.1, 6.6 Hz, 1H), 2.97 (dd, J=13.8, 7.8Hz, 1H), 2.44 (s, 3H) ppm.

Step F: Methyl3-benzyloxycarbonylamino-4-(2-methyl-5-nitro-phenyl)butanoate (3f)

Following the general procedure of Description 11 (Part C), methyl3-benzyloxycarbonylamino-4-(2-methyl-5-nitro-phenyl)butanoate (3f) isprepared from benzylN-[3-diazo-1-[(2-methyl-5-nitro-phenyl)methyl]-2-oxo-propyl]carbamate(3e) (2.5 g, 6.55 mmol) and a mixture of silver benzoate (AgBz) (0.75 g,3.3 mmol) in THF (5 mL) and triethylamine (TEA) (1.93 mL, 1.4 g, 13.1mmol) in a mixture of degassed anhydrous methanol (MeOH) (2.1 mL) anddegassed anhydrous tetrahydrofuran (THF) (15 mL) at room temperature andunder a nitrogen atmosphere. Evaporative work-up followed by silica gelcolumn chromatography purification (EtOAc/Hxn=2:3, v/v) afforded 2.1 g(82% yield) of the target compound (3f) as a colorless solid. R_(f)=0.33(EtOAc/Hxn=2:3 v/v). ¹H NMR (300 MHz, CDCl₃): δ 8.00-87.95 (m, 2H),7.38-7.24 (m, 6H), 5.48 (d, J=9.3 Hz, 1H), 5.02 (s, 2H), 4.30-4.21 (m,1H), 3.72 (s, 3H), 3.06-3.01 (m, 1H), 2.97-2.54 (m, 1H), 2.64-2.50 (m,2H), 2.48 (s, 3H) ppm.

Step G: Methyl4-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-butanoate (3g)

Following the General Procedure for of Description 6 (Variant A), methyl4-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-butanoate (3g) wasprepared from methyl3-benzyloxycarbonylamino-4-(2-methyl-5-nitro-phenyl)butanoate (3f) (2.1g, 5.4 mmol), iron powder (Fe) (2.7 g, 48.9 mmol), and calcium chloridedihydrate (CaCl₂.2H₂O) (0.35 g, 2.4 mmol) in a mixture of methanol(MeOH)/water (41 mL:7.5 mL, v/v). The reaction mixture was heated atreflux for about 2 hours (oil bath). Removal of the iron residues byfiltration and compound isolation procedures yielded 1.9 g(˜quantitative yield) of the target compound (3g) as a light yellowsolid which was of sufficient purity to be used directly in the neststep without further isolation and purification. ¹H NMR (300 MHz,DMSO-d⁶): δ 7.38-7.24 (m, 5H), 6.75 (d, J=7.5 Hz, 1H), 6.36-6.30 (m,2H), 4.97 (s, 2H), 4.72 (br. s, 2H), 4.15-3.85 (m, 1H), 3.50 (s, 3H),3.18-3.14 (m, 2H), 2.68-2.64 (m, 1H), 2.50-2.35 (m, 1H, superimposedwith solvent), 2.09 (s, 3H) ppm. LC/MS: R_(t)=1.158 min; ESI (pos.)m/z=379.1 (M+H⁺)⁺, 713.4 (2M+H⁺)⁺.

Step H: Methyl3-benzyloxycarbonylamino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoate(3h)

Following the General Procedure for of Description 7 (Variant A), methyl3-benzyloxycarbonylamino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoate(3i) was prepared from methyl4-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-butanoate (3h) (1.9g, 5.3 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (3.4 mL,26.5 mmol), and sodium cyanoborohydride (NaBH₃CN) (1.41 g of 95%purity=1.34 g, 21.3 mmol) in a mixture of methanol (MeOH) (34 mL) andtrifluoroacetic acid (TFA) (17 mL). Purification by silica gel columnchromatography with an ethyl acetate (EtOAc)/hexane mixture(EtOAc/hexane=1:2, v/v) afforded 2.16 g (85% yield) of the titlecompound (3h) as a colorless solid. R_(f)=0.37 (EtOAc/hexane=1:2, v/v,ninhydrine negative). ¹H NMR (300 MHz, CDCl₃): δ 7.36-7.24 (m, 5H), 7.03(d, J=8.4 Hz, 1H), 6.50 (dd, J=8.4, 2.7 Hz, 1H), 6.44-6.41 (br. m, 1H),5.50 (d, J=8.7 Hz, 1H), 5.08 (s, 2H), 4.26-418 (br. m, 1H), 3.70 (s,3H), 3.70-3.54 (m, 8H), 2.96 (dd, J=13.8, 6.3 Hz, 1H), 2.76 (dd, J=13.8,8.4 Hz, 1H), 2.55 (br. d, J=4.8 Hz, 2H), 2.26 (s, 3H) ppm. LC/MS:R_(t)=2.526 min; ESI (pos.) m/z=503.1 (M+H⁺)⁺. LC/UV: R_(t)=6.552 min,100.0% AUC at λ=254 nm.

Step I: 3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicAcid (3)

Following the General Procedure for of Description 8,3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid (3)was prepared through acidic hydrolysis of methyl3-benzyloxycarbonylamino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoate(3i) (2.16 g, 4.15 mmol) in a mixture of concentrated hydrochloric acid(HCl) (30 mL) and 1,4-dioxane (30 mL). The residue was purified bypreparative HPLC, immediately frozen after collection, followed byprimary lyophilization to afford 722 mg of the target compound (3) as acolorless powder. ¹H NMR (300 MHz, DMSO-d⁶): δ 7.30 (d, J=9.0 Hz, 1H),6.56-6.50 (m, 2H), 3.76-3.60 (br. m, 10H), 3.65-3.36 (br. m, 1H), 2.75(dd, J=13.5, 6.6 Hz, 1H), 2.65 (dd, J=13.2, 7.8 Hz, 1H), 2.13 (s, 3H),2.06 (d, J=3.9 Hz, 1H), 2.00 (dd, J=16.2, 9.3 Hz, 1H) ppm. LC/MS:R_(t)=1.094 min; ESI (pos.) m/z=333.1 (M+H⁺)⁺; ESI (neg.) m/z=330.9.0(M−H⁺)⁻. LC/UV: R_(t)=7.134 min, 95.5% AUC at λ=254 nm. The analyticaldata correspond to the analytical data of the (S)-isomer (5) and the(R)-isomer (6). Various batches of mono- or dihydrochloride salts of (3)can be prepared by primary lyophilization of solutions of (3) in aqueousacetonitrile (MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid(HCl) or an excess of 1.0 N or higher concentrated hydrochloric acid(HCl).

Example 43-Amino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic Acid (4)

Step A: 1-(Bromomethyl)-4-nitro-benzene (4a)

Following the General Procedure of Description 10,1-(bromomethyl)-4-nitro-benzene (4a) was prepared through bromination of(2-methyl-4-nitro-phenyl)methanol (2a) (18.0 g, 108 mmol) (prepared asdescribed in Example 2) dissolved in dichloromethane (DCM) (200 mL) witha solution of phosphorus tribromide (PBr₃) in (1.0 M PBr₃ in DCM) (108mL). Aqueous work-up yielded 16.0 g (64% yield) of a light yellow solidwhich was of sufficient purity to be used directly and without furtherisolation and purification in the next step. R_(f)=0.51 (EtOAc/Hxn=1:5v/v). The spectroscopic data correspond to the data provided in theliterature.

Step C: Methyl 2-amino-3-(2-methyl-4-nitro-phenyl)propanoateHydrochloride (4c)

Adapting literature protocols (methyl2-amino-3-(2-methyl-4-nitro-phenyl)propanoate Hydrochloride (4c) wasprepared through alkylation commercial methyl[(phenylmethylidene)amino]acetate (1.84 g, 10.4 mmol), with1-(bromomethyl)-4-nitro-benzene (4b) (2.86 g, 12.5 mmol), potassiumcarbonate (K₂CO₃) (4.31 g, 31.2 mmol), benzyltriethylammonium chloride(BTEAC) (237 mg, 1.04 mmol) in acetonitrile (MeCN) (30 mL). The reactionmixture was stirred for about 6 hours at room temperature, filtered, andconcentrated under reduced pressure using a rotary evaporator. Theresidue was diluted with diethyl ether (Et₂O) and the organic layer waswashed with brine. The phases were separated and the organic layer wasconcentrated to a total volume of about 20 mL. 1.0 M Hydrochloric acid(HCl) (50 mL) was added, and the reaction mixture was kept overnight atroom temperature. The reaction mixture was further diluted with diethylether (Et₂O) and the phases were separated. The aqueous phase wasconcentrated under reduced pressure using a rotary evaporator.

Following the General Synthesis of Description 4, the crude material wasdiluted with anhydrous methanol (MeOH) (20 mL) and treated with excessthionyl chloride (SOCl₂) at about 0° C. (ice bath). The reaction mixturewas subsequently heated to about 80° C. (oil bath) for about 1 h beforesolvents and volatiles were removed under reduced pressure using arotary evaporator to afford 2.18 g (76% yield) of the target compound(4c) as a colorless solid. LC/MS: R_(t)=0.687 min; ESI (pos.) m/z=239.1(M+H⁺)⁺.

Step D: Methyl2-benzyloxycarbonylamino-3-(2-methyl-4-nitro-phenyl)propanoate (4d)

Following the General Procedure of Description 5, methyl2-benzyloxycarbonylamino-3-(2-methyl-4-nitro-phenyl)propanoate (4d) wasprepared from methyl 2-amino-3-(2-methyl-4-nitro-phenyl)propanoatehydrochloride (4c) (2.18 g, 7.94 mmol), benzyl chloroformate (CbzCl,ZCl) (1.65 mL, 1.97 g, 11.9 mmol), and diisopropylethylamine (DIPEA,Hünigs-base) (3.92 mL, 3.07 g, 23.7 mmol) in dichloromethane (DCM) (50.0mL). Aqueous work-up and purification by silica gel colunchromatography(EtOAc/Hxn=1:2 v/v) afforded 1.94 g (40% yield) of the target compound(4d) as a colorless solid. R_(f)=0.44 (EtOAc/Hxn=1:2 v/v). ¹H NMR (400MHz, CDCl₃): δ 8.06-8.00 (m, 1H), 7.94-7.86 (m, 1H), 7.40-7.20 (m, 6H),5.36 (d, 1H), 5.06 (d, 1H), 5.00 (d, 1H), 4.70-4.60 (m, 1H), 3.68 (s,3H), 3.26 (dd, 1H), 3.04 (dd, 1H), 2.40 (s, 3H) ppm. LC/MS: R_(t)=2.085min; ESI (pos.) m/z=373.3 (M+H⁺)⁺; ESI (neg.) m/z=371.1 (M−H⁺)⁻.

Step E: 2-Benzyloxycarbonylamino-3-(2-methyl-4-nitro-phenyl)propanoicAcid (4e)

Adapting a literature protocol (Dayal, et al., Steroids, 1990, 55(5),233-237), a reaction mixture of methyl2-benzyloxycarbonylamino-3-(2-methyl-4-nitro-phenyl)propanoate (4d)(1.94 g, 5.20 mmol) and commercial lithium hydroxide monohydrate(LiOH.H₂O) (436 mg, 10.4 mmol) in a mixture of tetrahydrofuran(THF)/methanol (MeOH)/water (20:10:10 mL v/v/v) was stirred at roomtemperature. The reaction was followed by TLC and LC/MS to completion.Acidic aqueous work-up at about pH 4 and subsequent crystallization fromethyl acetate (EtOAc) furnished 900 mg (48% yield) of the targetcompound (4e) as a colorless solid. ¹H NMR (400 MHz, CDCl₃): δ 7.96-7.92(m, 1H), 7.90-7.80 (m, 1H), 7.36-7.18 (m, 6H), 5.62 (d, 1H), 5.00 (d,1H), 4.93 (d, 1H), 4.60-4.50 (m, 1H), 3.26 (dd, 1H), 2.98 (dd, 1H), 2.38(s, 3H) ppm. LC/MS: R_(t)=1.818 min; ESI (pos.) m/z=359.1 (M+H⁺)⁺; ESI(neg.) m/z=357.0 (M−H⁺)⁻.

Step F: BenzylN-[3-diazo-1-[(2-methyl-4-nitro-cyclohexa-2,4-dien-1-yl)methyl]-2-oxo-propyl]carbamate(4f)

Following the General Procedure of Description 12 (Parts A-B), benzylN-[3-diazo-1-[(2-methyl-4-nitro-cyclohexa-2,4-dien-1-yl)methyl]-2-oxo-propyl]carbamate(4f) was prepared from2-benzyloxycarbonylamino-3-(2-methyl-4-nitro-phenyl)propanoic acid (4e)(700 mg, 1.97 mmol), N-methylmorpholine (NMM) (433 μL, 398 mg, 3.94mmol), isobutyl chloroformate (515 μL, 538 mg, 3.94 mmol) in anhydroustetrahydrofuran (THF) (10 mL) and about 16 mmol of freshly prepareddiazomethane in Et₂O. Silica gel column chromatography (EtOAc/Hxn=1:2v/v) afforded 350 mg (46% yield) of the target compound (4f) as acolorless solid. R_(f)=0.24 (EtOAc/Hxn=1:2, v/v). ¹H NMR (400 MHz,CDCl₃): δ 8.02-7.98 (m, 1H), 7.96-7.88 (m, 1H), 7.38-7.20 (m, 6H), 5.40(d, 1H), 5.20 (s, 1H), 5.08 (d, 1H), 5.02 (d, 1H), 4.50-4.40 (m, 1H),3.18 (dd, 1H), 2.96 (dd, 1H), 2.42 (s, 3H) ppm. LC/MS: R_(t)=1.991 min;ESI (pos.) m/z=405.0 (M+Na⁺)⁺.

Step G: Methyl3-benzyloxycarbonylamino-4-(2-methyl-4-nitro-phenyl)butanoate (4g)

Following the General Procedure of Description 12 (Part C), methyl3-benzyloxycarbonylamino-4-(2-methyl-4-nitro-phenyl)butanoate (4g) wasprepared from benzylN-[3-diazo-1-[(2-methyl-4-nitro-cyclohexa-2,4-dien-1-yl)methyl]-2-oxo-propyl]carbamate(4f) (350 mg, 0.916 mmol) in Methanol (MeOH) (10 mL) and silver benzoate(AgBz) (0.75 g, 3.3 mmol) dissolved in triethylamine (TEA) (3.0 mL, 2.29g, 4.32 mmol). Silica gel column chromatography (EtOAc/Hxn=2:3 v/v)afforded 220 mg (62% yield) of the target compound (4g) as pale yellowsolid. ¹H NMR (400 MHz, CDCl₃): δ 8.02-7.98 (m, 1H), 7.92-7.86 (m, 1H),7.40-7.18 (m, 6H), 5.46 (d, 1H), 5.04-4.96 (m, 2H), 4.28-4.18 (m, 1H),3.69 (s, 3H), 3.08 (dd, 1H), 2.90 (dd, 1H), 2.60 (dd, 1H), 2.54 (dd,1H), 2.44 (s, 3H) ppm. LC/MS: R_(t)=2.082 min; ESI (pos.) m/z=387.2(M+H⁺)⁺; ESI (neg.) m/z=384.9 (M−H⁺)⁻.

Step H: Methyl4-(4-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-butanoate (4h)

Following the General Procedure for of Description 6 (Variant A), methyl4-(4-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-butanoate (4h) wasprepared from methyl3-benzyloxycarbonylamino-4-(2-methyl-4-nitro-phenyl)butanoate (4g) (220mg, 0.570 mmol), iron powder (Fe) (286 mg, 5.13 mmol), and anhydrouscalcium chloride (CaCl₂) (28 mg, 0.257 mmol) in 85 vol-% aqueousmethanol (MeOH) (20 mL). The reaction mixture was heated at reflux forabout 2 hours (oil bath). Removal of the iron residues by filtration andcompound isolation procedures yielded 200 mg (about quantitative yield)of the target compound (4h) as a light yellow oil which was ofsufficient purity to be used directly in the nest step without furtherisolation and purification. LC/MS: R_(t)=1.034 min; ESI (pos.) m/z=357.1(M+H⁺)⁺, 379.1 (M+Na⁺)⁺.

Step I: Methyl3-benzyloxycarbonylamino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoate(4i)

Following the General Procedure for of Description 7 (Variant A), methyl3-benzyloxycarbonylamino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoate(4i) was prepared from methyl4-(4-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-butanoate (4h) (200mg, 0.561 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (357μL, 2.87 mmol), and sodium cyanoborohydride (NaBH₃CN) (148 mg of 95%purity=141 mg, 2.24 mmol) in a mixture of methanol (MeOH) (20 mL) andtrifluoroacetic acid (TFA) (10 mL). Aqueous work-up and purification bysilica gel column chromatography with an ethyl acetate (EtOAc)/hexanemixture (EtOAc/hexane=2:3, v/v) afforded 260 mg (96% yield) of the titlecompound (4i) as a colorless oil. R_(f)=0.41 (EtOAc/Hxn=1:2, v/v). ¹HNMR (400 MHz, CDCl₃): δ 7.40-7.28 (m, 5H), 6.92-6.88 (d, 1H), 6.46-6.38(m, 2H), 5.38 (d, 1H), 5.10-5.00 (m, 2H), 4.10-4.00 (m, 1H), 3.70-3.56(m, 11H), 2.84 (dd, 1H), 2.70 (dd, 1H), 2.58-2.42 (m, 2H), 2.30 (s, 3H)ppm. LC/MS: R_(t)=2.470 min; ESI (pos.) m/z=481.2 (M+H⁺)⁺.

Step J: 3-Amino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicAcid (4)

Following the General Procedure for of Description 8,3-amino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoic acid (4)was prepared through hydrolysis of methyl3-benzyloxycarbonylamino-4-[4-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoate(4i) (260 mg, 0.54 mmol) in a mixture of concentrated hydrochloric acid(HCl) (1 mL) and 1,4-dioxane (1 mL). Purification by preparative HPLCafforded 82 mg (46% recovery) of the target compound (4) after primarylyophilization as a colorless solid. ¹H NMR (400 MHz, DMSO-d⁶): δ6.96-6.90 (d, 1H), 6.56-6.46 (m, 2H), 3.70-3.56 (br. m, 9H), 3.30 (br.s, superimposed with water signal, 3H), 2.70 (dd, 1H), 2.56 (dd, 1H),2.18 (s, 3H), 2.10-1.98 (m, 2H) ppm. LC/MS: R_(t)=1.195 min; ESI (pos.)m/z=333.1 (M+H⁺)⁺; ESI (neg.) m/z=331.0 (M−H⁺)⁻. LC/UV: R_(t)=7.896 min,96.5% AUC at λ=254 nm. Various batches of mono- or dihydrochloride saltsof (4) can be prepared by primary lyophilization of solutions of (4) inaqueous acetonitrile (MeCN) containing either 1.0 eq. of 1.0 Nhydrochloric acid (HCl) or an excess of 1.0 N or higher concentratedhydrochloric acid (HCl).

Example 5(3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicAcid (5)

Step A: O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-methyl(2R)-2-(tert-butoxycarbonylamino)-butanedioate (5a)

Following the General Procedure of Description 12,O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-methyl(2R)-2-(tert-butoxycarbonylamino)-butanedioate (5a) was prepared from(2R)-2-(tert-butoxycarbonylamino)-4-methoxy-4-oxo-butanoic acid (9.46 g,38.3 mmol) (commercially available or prepared from commercialH-D-Asp(OMe)-OH.HCl (10.5 g, 57.3 mmol) (preparable following theGeneral Procedure of Description 4) and Boc₂O (12.5 g, 57.3 mmol) in amixture of 1,4-dioxane (100 mL) and a freshly prepared 1.0 N aqueoussodium hydroxide (NaOH) solution (126 mL, 126 mmol) (9.46 g (67% yield)(Keller, et al., Org. Synth., 1985, 63, 160)), N-hydroxysuccinimide(1-hydroxypyrrolidine-2,5-dione, HOSu, NHS) (4.69 g, 40.8 mmol), anddicyclohexylcarbodiimide (DCC) (8.02 g, 38.9 mmol in ethyl acetate(EtoAc) (120 mL) at room temperature. Filtration and aqueous work-upfurnished 13.2 g (quantitative yield) of the title compound (5a) as acolorless solid which was of sufficient purity to be used directly andwithout further isolation and purification in the next step. R_(f)˜0.45(EtOAc/hexane=1:1, v/v). ¹H NMR (300 MHz, CDCl₃): δ 5.64 (br. d, J=9.3Hz, 1H), 5.03-4.96 (m, 1H), 3.75 (s, 3H), 3.12 (dd, J=17.4, 4.5 Hz, 1H),3.12 (dd, J=17.7, 4.5 Hz, 1H), 2.83 (br. s, 4H), 1.45 (s, 9H) ppm.LC/MS: R_(t)=1.463 min; ESI (pos.) m/z=367.15 (M+Na⁺)⁺.

Step B: Methyl (3R)-3-(tert-butoxycarbonylamino)-4-hydroxy-butanoate(5b)

Following the General Procedure of Description 13, methyl(3R)-3-(tert-butoxycarbonylamino)-4-hydroxy-butanoate (5b) was preparedthrough reduction of O¹-(2,5-dioxopyrrolidin-1-yl) O⁴-methyl(2R)-2-(tert-butoxycarbonylamino)-butanedioate (5a) (13.2 g, 38.3 mmol)with sodium borohydride (NaBH₄) (2.41 g, 63.7 mmol) in tetrahydrofuran(THF)/water (133 mL/17 mL). Aqueous work-up and purification by silicagel column chromatography with an ethyl acetate (EtOAc)/hexane mixture(EtOAc/hexane=4:3, v/v) furnished 5.73 g (43% yield over 3 steps) of thetitle compound (5b) as a colorless oil. R_(f)˜0.34 (EtOAc/hexane=1:1,v/v). ¹H NMR (400 MHz, CDCl₃): δ 5.30 (br. d, 1H), 4.06-3.92 (m, 1H),3.70-3.68 (m, superimposed, 5H), 2.63 (d, J=5.7 Hz, 2H), 1.43 (s, 9H)ppm. LC/MS: R_(t)=1.027 min; ESI (pos.) m/z=489.25 (2M+Na⁺)⁺. Theanalytical data correspond with the analytical data for the(S)-enatiomer in the literature (Dexter and Jackson, J. Org. Chem.,1999, 64, 7579-7585).

Step C: Methyl (3R)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (5c)

Following the General Procedure of Description 14, methyl(3R)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (5c) was preparedfrom methyl (3R)-3-(tert-butoxycarbonylamino)-4-hydroxy-butanoate (5b)(5.73 g, 24.6 mmol), iodine (I₂) (6.23 g, 24.6 mmol), triphenylphosphine(PPh₃) (6.45 g, 24.6 mmol), and imidazole (1.67 g, 24.6 mmol) inanhydrous dichloromethane (DCM) (100 mL). Aqueous reductive work-up andpurification by silica gel column chromatography with an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=7:3, v/v) furnished 4.30 g (51%yield) of the title compound (5c) as a colorless to beige solid.R_(f)˜0.79 (EtOAc/hexane=7:3, v/v). ¹H NMR (400 MHz, CDCl₃): δ 5.10 (br.d, J=7.2 Hz, 1H), 4.00-3.80 (m, 1H), 3.69 (s, 3H), 3.50-3.36 (m, 2H),2.76 (dd, J=16.5, 5.4 Hz, 1H), 2.62 (dd, J=16.5, 6.3 Hz, 1H), 1.43 (s,9H) ppm. The analytical data correspond with the analytical data for the(S)-enatiomer in the literature (Dexter and Jackson, J. Org. Chem.,1999, 64, 7579-7585).

Step D: Methyl(3S)-4-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)butanoate(5d)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (1.96 g, 30.0 mmol) was activated with elemental iodine (I₂) (190mg, 0.75 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (95μL, 81 mg, 0.75 mmol, 15 mol-%) in degassed anhydrousN,N-dimethylformamide (DMF) (6 mL). The zinc insertion product wasprepared from methyl (3R)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate(5c) (1.72 g, 5.0 mmol) in the presence of additional I₂ (190 mg, 0.75mmol, 15 mol-%) and TMSCl (95 μL, 81 mg, 0.75 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part B), the zincinsertion product of (5c) was used in situ to cross couple withcommercial 3-iodo-4-methyl-aniline (583 mg, 2.5 mmol) in the presence oftris(benzylideneacetone) dipalladium (Pd₂(dba)₃) (57 mg, 0.03 mmol, 2.5mol-%) and tris(o-tolyl)phosphine (P(o-tol)₃) (76 mg, 0.25 mmol, 10mol-%) in anhydrous degassed DMF (6 mL). Filtration, aqueous work-up,and purification by silica gel column chromatography with ethyl acetate(EtOAc)/hexane mixtures (EtOAc/hexane=7:3-1:1, v/v) furnished 1.04 g(65% yield) of the title compound (5d) as a yellow viscous oil.R_(f)˜0.28 (EtOAc/hexane=1:1, v/v). ¹H NMR (400 MHz, CDCl₃): δ 6.89 (d,J=8.4 Hz, 1H), 6.48-6.44 (m, 2H), 5.10-5.02 (br. m, 1H), 4.18-4.08 (m,1H), 3.65 (s, 3H), 3.30 (br. s, 2H), 2.82-2.78 (br. dd, 1H), 2.70 (dd,J=10.2, 6.0 Hz, 1H), 2.51 (dd, J=16.0, 5.2 Hz, 1H), 2.45 (dd, J=16.0,5.6 Hz, 1H), 2.19 (s, 3H), 1.38 (s, 9H) ppm. LC/MS: R_(t)=1.320 min.LC/MS: m/z=323.20 (M+H⁺)⁺, 345.15 (M+Na⁺)⁺.

Step E: Methyl(3S)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(5e)

Following the General Procedure of Description 7 (Variant C), methyl(3S)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(5e) was prepared from methyl(3S)-4-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(5d) (967 mg, 3.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (3.05 mL, 24.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (624 mgof 95% purity=593 mg, 9.43 mmol) in a mixture of methanol (MeOH) (18 mL)and 85 wt-% phosphoric acid (H₃PO₄) (8.1 mL). Aqueous work-up andpurification by silica gel column chromatography with an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=1:4, v/v) afforded 1.4 g (97%yield) of the title compound (5e) as a colorless oil. R_(f)˜0.32(EtOAc/Hxn=4:1, v/v). ¹H NMR (400 MHz, CDCl₃): δ 7.00 (d, J=8.5 Hz, 1H),6.49 (d, J=2.4 Hz, 1H), 6.42 (s, 1H), 5.10-5.04 (br. m, 1H), 3.69 (s,3H), 3.67-3.59 (m, 8H), 2.90-2.80 (m, 1H), 2.78-2.70 (m, 1H), 2.60-2.40(m, 2H), 2.23 (s, 3H), 1.37 (s, 9H) ppm. LC/MS: R_(t)=2.533 min; ESI(pos.) m/z=447.15 (M+H⁺)⁺, 469.15 (M+Na⁺)⁺.

Step F:(3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicAcid (5)

Following the General Procedure of Description 8,(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (5) was prepared through hydrolytic deprotection of methyl(3S)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(5e) (˜1.4 g, 3.13 mmol) in a mixture of concentrated hydrochloric acid(HCl) (7.5 mL) and 1,4-dioxane (7.5 mL). Part of the crude materialobtained after work-up was purified by preparative HPLC to afford ˜20 mgof the target compound (5) as a colorless solid after primarylyophilization. ¹H NMR (400 MHz, MeOH-d⁴): δ 7.04 (d, J=8.4 Hz, 1H),6.59 (d, J=8.4 Hz, 1H), 6.54 (s, 1H), 3.74-3.68 (br. m, 4H), 3.67-3.62(br. m, 4H), 3.58-3.50 (m, 1H), 2.92-2.86 (m, 2H), 2.44 (dd, J=16.8, 4.0Hz, 1H), 2.31 (dd, J=16.8, 8.4 Hz, 1H), 2.22 (s, 3H) ppm. The analyticaldata correspond to the analytical data obtained for racemic compound(3). Various batches of mono- or dihydrochloride salts of (6) can beprepared by primary lyophilization of solutions of (5) in aqueousacetonitrile (MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid(HCl) or an excess of 1.0 N or higher concentrated hydrochloric acid(HCl).

Example 6(3R)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicAcid (6)

Step A: O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-tert-butyl(2S)-2-(tert-butoxycarbonylamino)-butanedioate (6a)

Following the General Procedure of Description 12,O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-tert-butyl(2S)-2-(tert-butoxycarbonylamino)-butanedioate (6a) was prepared from(2S)-2-(tert-butoxycarbonylamino)-4-tert-butoxy-4-oxo-butanoic acid(8.32 g, 28.8 mmol) (commercially available or prepared following theGeneral Procedure of Description 4 from commercial H-L-Asp(OtBu)-OH(5.68 g, 30.0 mmol) and Boc₂O (6.55 g, 30.0 mmol) in a mixture of1,4-dioxane (25 mL) and a freshly prepared 1.0 N aqueous sodiumhydroxide (NaOH) solution (33 mL, 33 mmol) (8.33 g, 96% yield) (Keller,et al., Org. Synth., 1985, 63, 160)), N-hydroxysuccinimide(1-hydroxypyrrolidine-2,5-dione, HOSu, NHS) (3.53 g, 30.7 mmol), anddicyclohexylcarbodiimide (DCC) (6.03 g, 29.2 mmol in ethyl acetate(EtoAc) (100 mL) at room temperature. Filtration and aqueous work-upfurnished 11.8 g (quantitative yield) of the title compound (6a) as acolorless solid which was of sufficient purity to be used directly andwithout further isolation and purification in the next step. R_(f)˜0.56(EtOAc/hexane=1:1, v/v); R_(f)˜0.34 (EtOAc/hexane=1:2, v/v). ¹H NMR (300MHz, CDCl₃): δ 5.63 (d, J=9.3 Hz, 1H), 5.00-4.92 (m, 1H), 3.01 (dd,J=17.4, 5.1 Hz, 1H), 2.84 (dd, superimposed, J=17.4, 4.8 Hz, 1H), 2.84(s, superimposed, 4H), 1.47 (s, 9H), 1.45 (s, 9H) ppm. LC/MS:R_(t)=2.567 min; ESI (pos.) m/z=409.15 (M+Na⁺)⁺, 795.35 (2M+Na⁺)+; ESI(neg.) m/z=384.90.

Step B: tert-Butyl (3S)-3-(tert-butoxycarbonylamino)-4-hydroxy-butanoate(6b)

Following the General Procedure of Description 13, tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-hydroxy-butanoate (6b) was preparedthrough reduction of O¹-(2,5-dioxopyrrolidin-1-yl) O⁴-tert-butyl(2S)-2-(tert-butoxycarbonylamino)-butanedioate (6a) (11.8 g, 30.5 mmol)with sodium borohydride (NaBH₄) (2.31 g, 61.0 mmol) in tetrahydrofuran(THF)/water (110 mL/16 mL). Aqueous work-up and purification by silicagel column chromatography with an ethyl acetate (EtOAc)/hexane mixture(EtOAc/hexane=11:9, v/v) furnished 7.30 g (87% yield) of the titlecompound (6b) as a colorless viscous oil. R_(f)˜0.52 (EtOAc/hexane=1:1,v/v). ¹H NMR (400 MHz, CDCl₃): δ 5.23 (br. d, J=5.1 Hz, 1H), 4.02-3.90(m, 1H), 3.67 (d, J=4.8 Hz, 2H), 2.55 (dd, superimposed, J=15.3, 6.0 Hz,1H), 2.48 (dd, superimposed, J=15.3, 6.3 Hz, 1H), 1.44 (s, 9H), 1.43 (s,9H) ppm. LC/MS: R_(t)=1.887 min; ESI (pos.) m/z=298.10 (M+Na⁺)⁺;m/z=573.35 (2M+Na⁺)⁺.

Step C: tert-Butyl (3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate(6c)

Following the General Procedure of Description 14, tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) was preparedfrom tert-butyl (3S)-3-(tert-butoxycarbonylamino)-4-hydroxy-butanoate(6b) (4.46 g, 16.2 mmol), iodine (I₂) (4.10 g, 16.2 mmol),triphenylphosphine (PPh₃) (4.25 g, 16.2 mmol), and imidazole (1.10 g,16.2 mmol) in anhydrous dichloromethane (DCM) (70 mL). Aqueous reductivework-up and purification by silica gel column chromatography with ethylacetate (EtOAc)/hexane mixtures (EtOAc/hexane=7:3-1:1, v/v) furnished4.20 g (67% yield) of the title compound (6c) as a colorless to beigesolid. R_(f)˜0.79 (EtOAc/hexane=7:3, v/v). ¹H NMR (400 MHz, CDCl₃): δ5.09 (br. d, J=8.4 Hz, 1H), 3.90-3.80 (m, 1H), 3.44-3.30 (m, 2H), 2.60(dd, J=15.9, 6.0 Hz, 1H), 2.51 (dd, J=15.9, 6.0 Hz, 1H), 1.45 (s, 9H),1.43 (s, 9H) ppm. LC/MS: R_(t)=2.332 min; ESI (neg.) m/z=384.80 (M−H⁺)⁻.

Step D: tert-Butyl(3R)-4-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(6d)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (4.07 g, 62.3 mmol) is activated with elemental iodine (I₂) (396mg, 1.56 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl)(197 μL, 169 mg, 0.75 mmol, 15 mol-%) in degassed anhydrousN,N-dimethylformamide (DMF) (6 mL). The zinc insertion product wasprepared from tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) (4.01 g, 10.4mmol) in the presence of additional elemental 12 (396 mg, 1.56 mmol, 15mol-%) and TMSCl (197 μL, 169 mg, 0.75 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part B), The zincinsertion product of (6c) was used in situ to cross couple withcommercial 3-iodo-4-methyl-aniline (1.21 g, 5.2 mmol) in the presence oftris(benzylideneacetone) dipalladium (Pd₂(dba)₃) (119 mg, 0.13 mmol, 2.5mol-%) and tris(o-tolyl)phosphine (P(o-tol)₃) (158 mg, 0.52 mmol, 10mol-%) in anhydrous degassed DMF (6 mL). Filtration, aqueous work-up,and purification by silica gel column chromatography with an etlylacetate (EtOAc)/hexane mixture (EtOAc/hexane=7:3, v/v) furnished 1.15 g(61% yield) of the title compound (6d) as a yellow viscous oil.R_(f)˜0.28 (EtOAc/hexane=1:1, v/v). ¹H NMR (300 MHz, CDCl₃): δ 6.91 (d,J=8.1 Hz, 1H), 6.50-6.46 (m, 2H), 5.20-5.10 (br. m, 1H), 4.18-4.00 (m,1H), 3.24 (br. s, 2H), 2.88-2.78 (br. dd, 1H), 2.70 (dd, 1H), 2.44 (dd,J=15.4 Hz, 5.4 Hz, 1H), 2.36 (dd, J=15.4 Hz, 5.4 Hz, 1H), 2.22 (s, 3H),1.45 (s, 9H), 1.40 (s, 9H) ppm. LC/MS: R_(t)=1.433 min; ESI (pos.)m/z=365.20 (M+H⁺)⁺.

Step E: tert-Butyl(3R)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(6e)

Following the General Procedure of Description 7 (Variant C), tert-butyl(3R)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(6e) was prepared from tert-butyl(3R)-4-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(6d) (1.07 g, 2.92 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (3.0 mL, 23.6 mmol), and sodium cyanoborohydride (NaBH₃CN) (1.25 g of95% purity=1.19 g, 18.9 mmol) in a mixture of methanol (MeOH) (18 mL)and 85 wt-% phosphoric acid (H₃PO₄) (9 mL). Aqueous work-up andpurification by silica gel column chromatography with an ethyl acetate(EtOAc)/hexane mixture (EtOAc/hexane=1:6, v/v) afforded 1.06 g (74%yield) of the title compound (6e) as a colorless oil. R_(f)˜0.55(EtOAc/hexane=1:4, v/v). ¹H NMR (400 MHz, CDCl₃): δ 6.98 (d, J=8.4 Hz,1H), 6.45 (d, J=8.4 Hz, 1H), 6.42 (s, 1H), 5.00 (br. d, 1H), 4.18-4.00(m, 1H), 3.70-3.50 (m, 8H), 2.80-2.60 (m, 2H), 2.41 (dd, J=16.0, 5.6 Hz,1H), 2.32 (dd, J=16.0, 6.0 Hz, 1H), 2.21 (s, 3H), 1.42 (s, 9H), 1.32 (s,9H) ppm. LC/MS: R_(t)=2.944 min; ESI (pos.) m/z=489.20 (M+H⁺)⁺.

Step F:(3R)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicAcid (6)

Following the General Procedure of Description 8,(3R)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (6) was prepared through hydrolytic deprotection of tert-butyl(3R)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(6e) (160 mg, 0.33 mmol) in a mixture of concentrated hydrochloric acid(HCl) (1 mL) and 1,4-dioxane (1 mL). The crude material obtained afterwork-up was purified by preparative HPLC to afford ˜86 mg (79% recovery)of the target compound (6) as a colorless solid after primarylyophilization. ¹H NMR (400 MHz, MeOH-d⁴): δ 7.04 (d, J=8.4 Hz, 1H),6.59 (d, J=8.4 Hz, 1H), 6.54 (s, 1H), 3.74-3.68 (br. m, 4H), 3.67-3.62(br. m, 4H), 3.60-3.52 (m, 1H), 2.92-2.86 (m, 2H), 2.46 (dd, J=16.8, 4.0Hz, 1H), 2.34 (dd, J=16.8, 8.4 Hz, 1H), 2.22 (s, 3H) ppm. LC/MS:R_(t)=1.317 min; 100% AUC at λ=254 nm; ESI (pos.) m/z=333.05 (M+H⁺)⁺.LC/UV: R_(t)=8.489 min, 99.1% AUC at λ=254 nm. The analytical datacorrespond to the analytical data obtained for racemic compound (3).

Various batches of mono- or dihydrochloride salts of (6) can be preparedby primary lyophilization of solutions of (6) in aqueous acetonitrile(MeCN) containing either 1.0 eq. of 1.0 N hydrochloric acid (HCl) or anexcess of 1.0 N or higher concentrated hydrochloric acid (HCl).Following the General Procedure of Description 9 (Variant B),dihydrochloride salts of (6) can also be prepared through deprotectionwith 2 N HCl in diethyl ether (2 N HCl in Et₂O) to yield the targetcompound (6) as a solid dihydrochloride salt after evaporation of thesolvents and lyophilization from an aqueous solution. The material isgenerally of sufficient purity to be used directly and without furtherisolation and purification in in vitro and/or in vivo evaluation.

Example 7(3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoicAcid (7)

Step A: Methyl(3S)-4-(5-amino-2-methoxy-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(7a)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (392 mg, 6.0 mmol) was activated with elemental iodine (I₂) (38 mg,0.15 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (19 μL,16 mg, 0.15 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (2 mL). The zinc insertion product was prepared from methyl(3R)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (5c) (343 mg, 1.0mmol) in the presence of additional I₂ (38 mg, 0.15 mmol, 15 mol-%) andTMSCl (19 μL, 16 mg, 0.15 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part A), the zincinsertion product of (5c) was used in situ to cross couple withcommercial 3-iodo-4-methoxy-aniline (249 mg, 1.0 mmol) in the presenceof tris(benzylideneacetone) dipalladium (Pd₂(dba)₃) (23 mg, 0.025 mmol,2.5 mol-%) and tris(o-tolyl)phosphine (P(o-tol)₃) (30 mg, 0.10 mmol, 10mol-%) in anhydrous degassed DMF (3 mL). Filtration, aqueous work-up,and purification by silica gel column chromatography with ethyl acetate(EtOAc)/hexane and dichloromethane(DCM)/EtOAc mixtures(EtOAc/hexane=1:1, v/v→DCM/EtOAc=1:1, v/v) furnished ˜280 mg (66% yield;˜80% purity by AUC) of the title compound (7a) as a yellow viscous oil.R_(f)˜0.23 (EtOAc/hexane=1:1, v/v). ¹H NMR (300 MHz, CDCl₃): δ 6.90 (brs, 1H), 6.78 (br. d, J=8.1 Hz, 1H), 6.70 (d, J=8.7 Hz, 1H), 5.28 (br. d,J=8.1 Hz, 1H), 4.40-4.10 (m, 1H), 3.37 (s, 3H), 2.90-2.80 (br. m, 1H),2.75 (dd, J=12.6, 6.3 Hz, 1H), 2.50 (d, J=5.1 Hz, 2H), 1.35 (s, 9H) ppm.LC/MS: R_(t)=0.908 min; ESI (pos.) m/z=339.15 (M+H⁺)⁺, 677.40 (2M+H⁺)⁺,699.35 (2M+Na⁺)⁺.

Step B: Methyl(3S)-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-3-(tert-butoxycarbonylamino)butanoate(7b)

Following the General Procedure of Description 7 (Variant C), methyl(3S)-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-3-(tert-butoxycarbonylamino)butanoate(7b) was prepared from methyl(3S)-4-(5-amino-2-methoxy-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(7a) (280 mg, 0.83 mmol, ˜80% purity), 2-chloroacetaldehyde (˜50 wt-% inwater, ˜7.87 M) (842 μL, 6.63 mmol), and sodium cyanoborohydride(NaBH₃CN) (105 mg of 95% purity=100 mg, 1.59 mmol) in a mixture ofmethanol (MeOH) (5 mL) and 85 wt-% phosphoric acid (H₃PO₄) (2.5 mL).Aqueous work-up and purification by silica gel column chromatographywith an ethyl acetate (EtOAc)/hexane mixture (EtOAc/hexane=1:4, v/v)afforded 104 mg (27% yield) of the title compound (7b) as a colorlessoil. R_(f)˜0.30 (EtOAc/hexane=1:4); LC/MS: R_(t)=2.493 min; ESI (pos.)m/z=463.20 (M+H⁺)⁺.

Step C:(3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoicAcid (7)

Following the General Procedure of Description 8,(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]butanoicacid (7) was prepared from methyl(3S)-4-[5-[bis(2-chloroethyl)amino]-2-methoxy-phenyl]-3-(tert-butoxycarbonylamino)butanoate(7b) (104 mg, 0.224 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (3 mL) and 1,4-dioxane (3 mL) at about 60° C.(oil bath) for about 6 hours to afford ˜90 mg (˜95% yield) the titlecompound (7) as a dihydrochloride salt after evaporation of the solventsunder reduced pressure. LC/MS: R_(t)=1.207 min; ˜100% purity by AUC atλ=254 nm. ESI (pos.) m/z=349.05 (M+H⁺)⁺.

Example 8(3S)-3-Amino-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoicAcid (8)

Step A: Methyl(3S)-4-(3-amino-2,6-dimethyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(8a)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (392 mg, 6.0 mmol) is activated with elemental iodine (I₂) (38 mg,0.15 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (19 μL,16 mg, 0.15 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (3 mL). The zinc insertion product is prepared from methyl(3R)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (5c) (343 mg, 1.0mmol) in the presence of additional I₂ (38 mg, 0.15 mmol, 15 mol-%) andTMSCl (19 μL, 16 mg, 0.15 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part B), the zincinsertion product of (5c) is used in situ to cross couple with3-iodo-2,4-dimethyl-aniline (247 mg, 1.0 mmol; preparable followingDescription 6 from commercial 2-iodo-1,3-dimethyl-4-nitro-benzene (2.78g, 10.0 mmol), 5.6 g iron powder (Fe), and calcium chloride dehydrate(CaCl₂.2H₂O) (1.47 g, 10.0 mmol) in a mixture of ethanol (EtOH) (20 mL)and water (1 mL)) in the presence of tris(benzylideneacetone)dipalladium (Pd₂(dba)₃) (23 mg, 0.025 mmol, 2.5 mol-%) andtris(o-tolyl)phosphine (P(o-tol)₃) (30 mg, 0.10 mmol, 10 mol-%) inanhydrous degassed DMF (3 mL). Filtration, aqueous work-up, andpurification by silica gel column chromatography furnish the titlecompound (8a).

Step B: Methyl(3S)-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(8b)

Following the General Procedure of Description 7 (Variant C), methyl(3S)-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(8b) is prepared from methyl(3S)-4-(3-amino-2,6-dimethyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(8a) (336 mg, 1.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (700 μL, 5.51 mmol), and sodium cyanoborohydride (NaBH₃CN) (264 mg of95% purity=251 mg, 4.0 mmol) in a mixture of methanol (MeOH) (6 mL) and85 wt-% phosphoric acid (H₃PO₄) (3 mL). Aqueous work-up and purificationby silica gel column chromatography furnish the title compound (8b).

Step C:(3S)-3-Amino-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoicAcid (8)

Following the General Procedure of Description 8,(3S)-3-amino-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]butanoicacid (8) is prepared from methyl(3S)-4-[3-[bis(2-chloroethyl)amino]-2,6-dimethyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(8b) (461 mg, 1.0 mmol) by hydrolysis in a mixture of concentratedhydrochloric acid (HCl) (about 5 mL) and 1,4-dioxane (about 5 mL) atabout 60° C. for about 15 hours to afford the title compound (8) as asolid dihydrochloride salt after isolation using evaporation andlyophilization. The material thus obtained is purified by preparativeRP-HPLC using a water/acetonitrile/0.1 vol-% formic acid gradient toafford the title compound (8) as a dihydrochloride salt after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl).

Example 9(3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicAcid (9)

Step A: O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-methyl(2R)-2-benzyloxycarbonylamino-2-methyl-butanedioate (9a)

Following the General Procedure of Description 12,O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-methyl(2R)-2-benzyloxycarbonylamino-2-methyl-butanedioate (9a) is preparedfrom (2R)-2-benzyloxycarbonylamino-4-methoxy-2-methyl-4-oxo-butanoicacid (2.95 g, 10.0 mmol) (preparable in two steps from commercial(R)-α-methyl aspartic acid: i) SOCl₂, MeOH, 0° C.→room temperature, 3 h;ii) Cbz-OSu (N-(benzyloxycarbonyloxy)succinimide), aq. K₃PO₄/toluene, 0°C.→room temperature, 14 h) following a literature known protocol(Gauvreau, et al., International Publication No. WO 2008/088690)),N-hydroxysuccinimide (1-hydroxypyrrolidine-2,5-dione, HOSu, NHS) (1.21g, 10.5 mmol), and dicyclohexylcarbodiimide (DCC) (2.06 g, 10.0 mmol inethyl acetate (EtoAc) (40 mL) at room temperature. Filtration andaqueous work-up furnish the title compound (9a) which may be ofsufficient purity to be used directly in the next step without furtherisolation and purification.

Step B: Methyl(3R)-3-benzyloxycarbonylamino-4-hydroxy-3-methyl-butanoate (9b)

Following the General Procedure of Description 13, methyl(3R)-3-benzyloxycarbonylamino-4-hydroxy-3-methyl-butanoate (9b) isprepared through reduction of O¹-(2,5-Dioxopyrrolidin-1-yl) O⁴-methyl(2R)-2-benzyloxycarbonylamino-2-methyl-butanedioate (9a) (3.92 g, 10.0mmol) with sodium borohydride (NaBH₄) (757 mg, 20.0 mmol) intetrahydrofuran (THF)/water (40 mL/5 mL). Aqueous work-up andpurification by silica gel column chromatography furnish the titlecompound (9b).

Step C: Methyl (3R)-3-benzyloxycarbonylamino-4-iodo-3-methyl-butanoate(9c)

Following the General Procedure of Description 14, methyl(3R)-3-benzyloxycarbonylamino-4-iodo-3-methyl-butanoate (9c) is preparedfrom methyl (3R)-3-benzyloxycarbonylamino-4-hydroxy-3-methyl-butanoate(9b) (2.81 g, 10.0 mmol), iodine (I₂) (2.54 g, 10.0 mmol),triphenylphosphine (PPh₃) (2.62 g, 10.0 mmol), and imidazole (681 mg,10.0 mmol) in anhydrous dichloromethane (DCM) (50 mL). Aqueous reductivework-up and purification by silica gel column chromatography furnish thetitle compound (9c).

Step D: Methyl(3S)-4-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-3-methyl-butanoate(9d)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (784 mg, 12.0 mmol) is activated with elemental iodine (I₂) (76 mg,0.30 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (38 μL,32 mg, 0.30 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (6 mL). The zinc insertion product is prepared from methyl(3R)-3-benzyloxycarbonylamino-4-iodo-3-methyl-butanoate (9c) (782 mg,2.0 mmol) in the presence of additional I₂ (76 mg, 0.30 mmol, 15 mol-%)and TMSCl (38 μL, 32 mg, 0.30 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part B), the zincinsertion product of (9c) is used in situ to cross couple withcommercial 3-iodo-4-methyl-aniline (466 mg, 2.0 mmol) in the presence oftris(benzylideneacetone) dipalladium (Pd₂(dba)₃) (46 mg, 0.05 mmol, 2.5mol-%) and tris(o-tolyl)phosphine (P(o-tol)₃) (60 mg, 0.20 mmol, 10mol-%) in anhydrous degassed DMF (6 mL). Filtration, aqueous work-up,and purification by silica gel column chromatography furnish the titlecompound (9d).

Step E: Methyl(3S)-3-benzyloxycarbonylamino-4-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-3-methyl-butanoate(9e)

Following General Procedure of Description 16, methyl(3S)-3-benzyloxycarbonylamino-4-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-3-methyl-butanoate(9e) is prepared from methyl(3S)-4-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-3-methyl-butanoate(9d) (3.70 g, 10.0 mmol) through reaction with ethylene oxide (12.5 mL,11.0 g, 100.0 mmol) in 15 mL of 50 vol.-% aqueous acetic acid (HOAc) for24 hours at room temperature to yield the title compound (9e) afteraqueous work-up and purification by silica gel chromatography.

Step F: Methyl(3S)-3-benzyloxycarbonylamino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoate(9f)

Following the General Procedure of Description 17, methyl(3S)-3-benzyloxycarbonylamino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoate(9f) is prepared from methyl(3S)-4-(5-amino-2-methyl-phenyl)-3-benzyloxycarbonylamino-3-methyl-butanoate(9d) (1.85 g, 5.0 mmol) through reaction with i) thionyl chloride(SOCl₂) (3.63 mL, 5.93 g, 50 mmol) in 25 mL of anhydrous chloroform(CHCl₃) for 2 hours at reflux temperature (Variant A), ii) phosphorylchloride (POCl₃) (2.34 mL, 3.83 g, 25.0 mmol) in anhydrous benzene (20mL) for about 5 h at a temperature of about 80° C. (Variant B), iii)methanesulfonyl chloride (MsCl) (1.94 mL, 2.86 g, 25.0 mmol) inanhydrous pyridine (20 mL) for 2 hours at 90° C. (Variant C), or iv)triphenylphosphine (Ph₃P) (2.62 g, 10.0 mmol) and carbon tetrachloride(CCl₄) (1.45 mL, 2.31 g, 15.0 mmol) in anhydrous dichloromethane (DCM)(20 mL) at room temperature for 8 hours (Variant D) to yield the targetcompound (9f) after work-up and purification by silica gel columnchromatography.

Step G:(3S)-3-Amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicAcid (9)

Following the General Procedure of Description 8,(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoicacid (9) is prepared through hydrolytic deprotection of methyl(3S)-3-benzyloxycarbonylamino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-methyl-butanoate(9f) (495 mg, 1.0 mmol) in a mixture of concentrated hydrochloric acid(HCl) (5 mL) and 1,4-dioxane (5 mL) and obtained as a soliddihydrochloride salt after isolation using evaporation andlyophilization. The material thus obtained is purified by preparativeRP-HPLC using a water/acetonitrile/0.1 vol-% formic acid gradient toafford the title compound (9) as a dihydrochloride salt after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl).

Example 10[(2R)-2-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicAcid (10)

Step A: Methyl(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)-phosphoryl)propanoate(10a)

Adapting literature protocols, methyl(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)-phosphoryl)propanoate(10a) is prepared form commercial methyl(2R)-2-(tert-butoxycarbonylamino)-3-iodo-propanoate (Jackson andPerez-Gonzalez, Org. Synth., 2005, 81, 77-88) (3.29 g, 10.0 mmol) and1-(1-ethoxy-1-ethoxyphosphonoyl-ethoxy)ethane (2.10 g, 10.0 mmol)(preparable from 80-90 wt-% aqueous hypophosphorous acid (H₃PO₂),triethylorthoacetate and BF₃-etherate (BF₃.OEt₂) catalyst; (Baylis,Tetrahedron Lett., 1995, 36(51), 9385-9388) in the presence of sodiumhydride (NaH) (60 wt-% suspension in mineral oil) (400 mg, 10.0 mmol) inanhydrous toluene (50 mL). The reaction if followed by TLC and/or LC/MSto completion. Aqueous work-up and purification by silica gel columnchromatography furnish the title compound (10a).

Step B:(2R)-2-(tert-Butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)-phosphoryl)propanoicAcid (10b)

Adapting a literature known protocol (Dayal, et al., Steroids, 1990,55(5), 233-237), a reaction mixture of methyl(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)-phosphoryl)propanoate(10a) (4.11 g, 10.0 mmol) and commercial lithium hydroxide monohydrate(LiOH.H₂O) (839 mg, 20.0 mmol) in a mixture of water (20 mL) andmethanol (MeOH) (5 mL) is stirred at room temperature. The reaction ismonitored by TLC and/or LC/MS to completion. Acidic aqueous work-up andpurification by silica gel column chromatography furnish the titlecompound(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)propanoicacid (10b) which may be used directly in the next step without furtherisolation and purification.

Step C: (2,5-Dioxopyrrolidin-1-yl)(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)propanoate(10c)

Following the General Procedure of Description 12,(2,5-dioxopyrrolidin-1-yl)(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)propanoate(10c) is prepared from(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)-phosphoryl)propanoicacid (10b) (3.97 g, 10.0 mmol), N-hydroxysuccinimide(1-hydroxypyrrolidine-2,5-dione, HOSu, NHS) (1.21 g, 10.5 mmol), anddicyclohexylcarbodiimide (DCC) (2.06 g, 10.0 mmol in ethyl acetate(EtoAc) (40 mL) at room temperature. Filtration and aqueous work-upfurnish the title compound (10c) which may be of sufficient purity to beused directly in the next step without further isolation andpurification.

Step D: tert-ButylN-[(1R)-1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-hydroxy-ethyl]carbamate(10d)

Following the General Procedure of Description 13, tert-butylN-[(1R)-1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-hydroxy-ethyl]carbamate(10d) is prepared through reduction of (2,5-dioxopyrrolidin-1-yl)(2R)-2-(tert-butoxycarbonylamino)-3-(1,1-diethoxyethyl(ethoxy)phosphoryl)propanoate(10c) (4.95 g, 10.0 mmol) with sodium borohydride (NaBH₄) (757 mg, 20.0mmol) in tetrahydrofuran (THF)/water (40 mL/5 mL). Aqueous work-up andpurification by silica gel column chromatography furnish the titlecompound (10d).

Step E: tert-ButylN-[(1S)-1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-iodo-ethyl]carbamate(10e)

Following the General Procedure of Description 14, tert-butylN-[(1S)-1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-iodo-ethyl]carbamate(10e) is prepared from tert-butylN-[(1R)-1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-hydroxy-ethyl]carbamate(10d) (3.83 g, 10.0 mmol), iodine (I₂) (2.54 g, 10.0 mmol),triphenylphosphine (PPh₃) (2.62 g, 10.0 mmol), and imidazole (681 mg,10.0 mmol) in anhydrous dichloromethane (DCM) (50 mL). Aqueous reductivework-up and purification by silica gel column chromatography furnish thetitle compound (10e).

Step F: tert-ButylN-[(1R)-1-[(5-amino-2-methyl-phenyl)methyl]-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]carbamate(10f)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (784 mg, 12.0 mmol) is activated with elemental iodine (I₂) (76 mg,0.30 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (38 μL,32 mg, 0.30 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (6 mL). The zinc insertion product is prepared from tert-butylN-[(1S)-1-[(1,1-diethoxyethyl(ethoxy)phosphoryl)methyl]-2-iodo-ethyl]carbamate(10e) (987 mg, 2.0 mmol) in the presence of additional I₂ (76 mg, 0.30mmol, 15 mol-%) and TMSCl (38 μL, 32 mg, 0.30 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part B), which isused in situ to cross couple with commercial 3-iodo-4-methyl-aniline(466 mg, 2.0 mmol) in the presence of tris(benzylideneacetone)dipalladium (Pd₂(dba)₃) (46 mg, 0.05 mmol, 2.5 mol-%) andtris(o-tolyl)phosphine (P(o-tol)₃) (60 mg, 0.20 mmol, 10 mol-%) inanhydrous degassed DMF (6 mL). Filtration, aqueous work-up, andpurification by silica gel column chromatography furnish the titlecompound (10f).

Step G: tert-ButylN-[(1R)-1-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]methyl]-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]carbamate(10g)

Following the General Procedure of Description 7 (Variant C), tert-butylN-[(1R)-1-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]methyl]-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]carbamate(10g) is prepared from tert-butylN-[(1R)-1-[(5-amino-2-methyl-phenyl)methyl]-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]carbamate(10f) (472 mg, 1.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (700 μL, 5.51 mmol), and sodium cyanoborohydride (NaBH₃CN) (264 mg of95% purity=251 mg, 4.0 mmol) in a mixture of methanol (MeOH) (6 mL) and85 wt-% phosphoric acid (H₃PO₄) (3 mL). Aqueous work-up and purificationby silica gel column chromatography furnish the title compound (10g).

Step H:[(2R)-2-Amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicAcid (10)

Following the General Procedure of Description 8,[(2R)-2-amino-3-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]propyl]phosphinicacid (10) is prepared through hydrolytic deprotection of tert-butylN-[(1R)-1-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]methyl]-2-(1,1-diethoxyethyl(ethoxy)phosphoryl)ethyl]carbamate(10g) (598 mg, 1.0 mmol) in a mixture of concentrated hydrochloric acid(HCl) (5 mL) and 1,4-dioxane (5 mL) and obtained as a soliddihydrochloride salt after isolation using evaporation andlyophilization. The material thus obtained is purified by preparativeRP-HPLC using a water/acetonitrile/0.1 vol-% formic acid gradient toafford the title compound (9) as a dihydrochloride salt after finallyophilization of the solvents in the presence of an excess of 1.0 Mhydrochloric acid (HCl).

Example 11(3R)-3-Amino-4-[5-(2-methylsulfonyloxyethyl(propyl)amino)-2-methyl-phenyl]butanoicAcid (11)

Step A: tert-Butyl(3R)-4-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(1a)

Variant A:

Following General Procedure of Description 16, tert-butyl(3R)-4-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11a) is prepared from tert-butyl(3R)-4-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(6d) (3.64 g, 10.0 mmol) through reaction with ethylene oxide (12.5 mL,11.0 g, 100.0 mmol) in 15 mL of 50 vol.-% aqueous acetic acid (HOAc) for24 hours at room temperature to yield the title compound (11) afteraqueous work-up and purification by silica gel chromatography.

Variant B:

Adapting literature known protocols (Palmer, et al., J. Med. Chem. 1990,33(1), 112-121; Coggiola, et al., Bioorg. Med. Chem. Lett., 2005,15(15), 3551-3554; Verny and Nicolas, J. Label. Cmpds Radiopharm., 1988,25(9), 949-955; and Lin, Bioorg. Med. Chem. Lett., 2011, 21(3),940-943), a reaction mixture of tert-butyl(3R)-4-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-butanoate(6d) (3.64 g, 10.0 mmol) and commercial 2-chloroethanol (2.68 mL, 3.22g, 40.0 mmol), calcium carbonate (CaCO₃) (2.0 g, 20.0 mmol, 2.0equivalents) in water (about 35 mL), and a catalytic amount of potassiumiodide (KI) (166 mg, 1.0 mmol, 10 mol-%) is heated at reflux for about12-24 hours. The reaction is followed by TLC and/or LC/MS to completion.The pH of the reaction mixture is adjusted to ˜7 with a 2.5 M (10 wt-%)aqueous solution of sodium hydroxide (NaOH). Aqueous work-up andpurification by silica gel column chromatography furnish the targetcompound (11a).

Step B: tert-Butyl(3R)-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11b)

Following the General Procedures of Description 18, tert-butyl(3R)-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11b) is prepared from tert-butyl(3R)-4-[5-(bis(2-hydroxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11a) (2.26 g, 5.0 mmol) and either methanesulfonyl anhydride (Ms₂O)(3.48 g, 20.0 mmol) in the presence of triethylamine (TEA, Et₃N) (3.48mL, 2.54 g, 25.0 mmol) and 4-N,N-(dimethylamino)pyridine (DMAP) (122 mg,1.0 mmol, 20 mol-%) in anhydrous dichloromethane (DCM) (30 mL) (VariantA) or methanesulfonyl chloride (MsCl) (0.96 mL, 1.44 g, 12.5 mmol) inthe presence of triethylamine (TEA, Et₃N) (2.10 mL, 1.52 g, 15.0 mmol)or pyridine (4.0 mL, 3.96 g, 50.0 mmol) in anhydrous dichloromethane(DCM) (30 mL) (Variant B) to yield the target compound (11b) afteraqueous work-up and purification by silica gel column chromatography.

Step C:(3R)-3-Amino-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicAcid (11)

Following the General Procedure of Description 9 (Variant B),(3R)-3-amino-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (11) is prepared from tert-butyl(3R)-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11b) (609 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (11) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 12(3R)-3-Amino-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic Acid(12)

Step A: tert-Butyl(3R)-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(12a)

Following the General Procedure of Description 19, tert-butyl(3R)-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(12a) is prepared from tert-butyl (3R)-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11b) (1.22 g, 2.0 mmol) through reaction with lithium bromide (LiBr)(1.74 g, 20.0 mmol) in tetrahydrofuran (THF) (10 mL) at refluxtemperature for about 6 h to yield the title compound (12a) afteraqueous work-up and purification by silica gel column chromatographywith ethyl acetate (EtOAc) and hexane mixtures.

Step B:(3R)-3-Amino-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic Acid(12)

Following the General Procedure of Description 9 (Variant A),(3R)-3-amino-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]butanoic acid(12) is prepared from tert-butyl(3R)-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(12a) (578 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 h to yield the target compound (12) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution. The material may be further purified bypreparative RP-HPLC using a water/acetonitrile/0.1 vol-% formic acidgradient followed by lyophilization in the presence of 1.0 equivalent oran excess of 1.0 M hydrobromic acid (HBr).

Example 13(3R)-3-Amino-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicAcid (13)

Step A: tert-Butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(13a)

Following the General Procedure of Description 19, tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(13a) is prepared from tert-butyl(3R)-4-[5-(bis(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(11b) (2.44 g, 4.0 mmol) through reaction with lithium chloride (LiCl)(186 mg, 2.2 mmol) in anhydrous acetonitrile (MeCN) (20 mL) at refluxtemperature for 1.5 h to yield the title compound (13a) after aqueouswork-up and purification by silica gel column chromatography.

Step B:(3R)-3-Amino-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicAcid (13)

Following the General Procedure of Description 9 (Variant A),(3R)-3-amino-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (13) is prepared from tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(13a) (549 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 h to yield the target compound (13) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution.

Example 14(3R)-3-Amino-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoicAcid (14)

Step A: tert-Butyl(3R)-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(14a)

Following the General Procedure of Description 19, tert-butyl(3R)-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(14a) is prepared from tert-Butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoate(13a) (1.10 g, 2.0 mmol) through reaction with lithium chloride (LiBr)(191 mg, 2.2 mmol) in anhydrous acetonitrile (MeCN) (10 mL) at refluxtemperature for about 2 h to yield the title compound (14a) afteraqueous work-up and purification by silica gel column chromatography.

Step B:(3R)-3-Amino-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoicAcid (14)

Following the General Procedure of Description 9 (Variant A),(3R)-3-amino-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]butanoicacid (14) is prepared from tert-butyl(3R)-4-[5-(2-bromoethyl(2-chloroethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(14a) (533 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 h to yield the target compound (14) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution. The material may be further purified bypreparative RP-HPLC followed using a water/acetonitrile/0.1 vol-% formicacid gradient followed by lyophilization.

Example 15(3R)-3-Amino-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicAcid (15)

Step A: tert-Butyl(3R)-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(15a)

Adapting literature known protocols (Emmons and A. F. Ferris, J. AmChem. Soc. 1953, 75(9), 2257-2257), tert-butyl(3R)-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(15a) is prepared from tert-butyl(3R)-4-[5-(bis(2-bromoethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(12a) (1.16 g, 2.0 mmol) with silver methanesulfonate (silver mesylate,AgOMs) (365 mg, 1.8 mmol) in anhydrous acetonitrile (MeCN) (8 mL) atreflux temperature for about 1 h under exclusion of light. Aqueouswork-up and purification by silica gel column chromatography afford thetitle compound (15a).

Step B:(3R)-3-Amino-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicAcid (15)

Following the General Procedure of Description 9 (Variant A),(3R)-3-amino-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]butanoicacid (15) is prepared from tert-butyl(3R)-4-[5-(2-bromoethyl(2-methylsulfonyloxyethyl)amino)-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(15a) (594 mg, 1.0 mmol) through deprotection in a trifluoroacetic acid(TFA)/dichloromethane (DCM) mixture (TFA/DCM=1:1 v/v, 10 mL) at roomtemperature for about 6 h to yield the target compound (15) as aditrifluoroacetate salt after evaporation and lyophilization from anaqueous acetonitrile solution. The material may be further purified bypreparative RP-HPLC followed using a water/acetonitrile/0.1 vol-% formicacid gradient followed by lyophilization.

Example 16(3S)-3-Amino-4-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]amino]-4-oxo-butanoicAcid (16)

Step A: N-[5-[Bis(2-chloroethyl)amino]-2-methyl-phenyl]acetamide (16a)

Following the General Procedure of Description 7 (Variant A),N-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]acetamide (16a) isprepared from commercial N-(5-amino-2-methylphenyl)acetamide (161 mg,1.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (700 μL,5.51 mmol), and sodium cyanoborohydride (NaBH₃CN) (264 mg of 95%purity=251 mg, 4.0 mmol) in a mixture of methanol (MeOH) (6 mL) andtrifluoroacetic acid (3 mL). Aqueous work-up and purification by silicagel column chromatography furnish the title compound (16a).

Step B: N¹,N¹-Bis(2-chloroethyl)-4-methyl-benzene-1,3-diamine (16b)

Following the General Procedure of Description 8,N¹,N¹-bis(2-chloroethyl)-4-methyl-benzene-1,3-diamine (16b) is preparedfrom methyl N-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]acetamide(16a) (289 mg, 1.0 mmol) by hydrolysis in concentrated hydrochloric acid(HCl) (about 5 mL) at reflux for about 2 hours to afford the titlecompound (16b) as a solid dihydrochloride salt after isolation usingevaporation and lyophilization. The material thus obtained can be useddirectly in the nest step without further isolation and purification inthe next step.

Step C: tert-Butyl(3S)-4-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]amino]-3-(tert-butoxycarbonylamino)-4-oxo-butanoate(16c)

Adapting a literature known protocol (Levi and Weed, U.S. Pat. No.3,235,594 (1966)), to a solution of O¹-(2,5-Dioxopyrrolidin-1-yl)O⁴-tert-butyl (2S)-2-(tert-butoxycarbonylamino)-butanedioate(Boc-L-Asp(Osu)-OtBu) (6a) (386 mg, 1.0 mmol) in anhydrous acetonitrile(MeCN) (10 mL) is addedN¹,N¹-bis(2-chloroethyl)-4-methyl-benzene-1,3-diamine (16b) as a bishydrochloride salt (320 mg, 1.0 mmol) followed by neat triethylamine(Et₃N, TEA) (321 μL, 233 mg, 2.3 mmol). The reaction mixture is stirredfor about 12 h at room temperature. The reaction is followed by TLCand/or LC/MS to completion. The volatile solvents are removed underreduced pressure using a rotary evaporator. Aqueous work-up andpurification by silica gel column chromatography furnish the targetcompound (16c).

Step D:(3S)-3-Amino-4-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]amino]-4-oxo-butanoicAcid (16)

Following the General Procedure of Description 9 (Variant B),(3S)-3-amino-4-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]amino]-4-oxo-butanoicacid (16) is prepared from tert-butyl(3S)-4-[[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]amino]-3-(tert-butoxycarbonylamino)-4-oxo-butanoate(16c) (518 mg, 1.0 mmol) in 2.0 N HCl in diethyl ether (2.0 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (15) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent of 1.0 Mhydrochloric acid (HCl).

Example 17 (3S)-3-Amino-4-[2-[bis(2-chloroethyl)amino]phenoxy]butanoicAcid (17)

Step A: tert-Butyl(3S)-3-(tert-butoxycarbonylamino)-4-(2-nitrophenoxy)butanoate (17a)

Adapting literature procedures (Bookster, et al., InternationalPublication No. WO 2010/047982), tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-(2-nitrophenoxy)butanoate (17a) isprepared from tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) (1.16 g, 3.0mmol) and commercial 2-nitrophenol (558 mg, 4.0 mmol) in the presence ofcesium carbonate (Cs₂CO₃) (1.63 g, 5.0 mmol) in anhydrousN,N-dimethylformamide (DMF) (10 mL) at 50° C. (oil bath). Aqueouswork-up and purification by silica gel chromatography furnish the titlecompound (17a).

Step B: tert-Butyl(3S)-4-(2-aminophenoxy)-3-(tert-butoxycarbonylamino)butanoate (17b)

Following the General Procedure of Description 6 (Variant B), tert-butyl(3S)-4-(2-aminophenoxy)-3-(tert-butoxycarbonylamino)butanoate (17b) isprepared by catalytic reduction of tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-(2-nitrophenoxy)butanoate (17a) (793mg, 2.0 mmol) in the presence of 10 wt-% palladium on charcoal (Pd/C)containing ˜50 wt-% water (˜350 mg) in methanol (MeOH) (20 mL) and underan atmosphere of hydrogen (˜15 psi, H₂-balloon). The crude material,after filtration over Celite® 545, may be used directly and withoutfurther isolation in the next step or may be purified by silica gelchromatography to afford the title compound (17b).

Step C: tert-Butyl(3S)-4-[2-[bis(2-chloroethyl)amino]phenoxy]-3-(tert-butoxycarbonyl-amino)butanoate(17c)

Following the General Procedure of Description 7 (Variant C), tert-butyl(3S)-4-[2-[bis(2-chloroethyl)amino]phenoxy]-3-(tert-butoxycarbonyl-amino)butanoate(17c) is prepared from tert-butyl(3S)-4-(2-aminophenoxy)-3-(tert-butoxycarbonylamino)butanoate (17b) (733mg, 2.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87 M) (1.27mL, 10.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (529 mg of 95%purity=503 mg, 8.0 mmol) in a mixture of methanol (MeOH) (10 mL) and 85wt-% phosphoric acid (H₃PO₄) (3 mL). Aqueous work-up and purification bysilica gel column chromatography furnish the title compound (17c).

Step D: (3S)-3-Amino-4-[2-[bis(2-chloroethyl)amino]phenoxy]butanoic Acid(17)

Following the General Procedure of Description 9 (Variant B),(3S)-3-amino-4-[2-[bis(2-chloroethyl)amino]phenoxy]butanoic acid (17) isprepared from tert-butyl(3S)-4-[2-[bis(2-chloroethyl)amino]phenoxy]-3-(tert-butoxycarbonyl-amino)butanoate(17c) (491 mg, 1.0 mmol) in 2.0 N HCl in diethyl ether (2.0 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (17) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent of 1.0 Mhydrochloric acid (HCl).

Example 18(3R)-3-Amino-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]pentanoicAcid (18)

Step A:[(2S)-4-tert-Butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]-triphenyl-phosphoniumIodide (18a)

Adapting a literature procedure (Rémond, et al, J. Org. Chem., J. Org.Chem. 2012, 77, 7579-7587),[(2S)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]-triphenyl-phosphoniumiodide (18a) is prepared from tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) (1.16 g, 3.0mmol) and triphenylphosphine (Ph₃P) (1.81 g, 6.9 mmol) at 80° C. under anitrogen atmosphere. After cooling to room temperature and tritruationwith toluene and diethyl ether (Et₂O), the residue is purified by silicagel column chromatography with an acetone and hexane mixture.

Step B: tert-Butyl(3S)-3-(tert-butoxycarbonylamino)-5-(2-methyl-5-nitro-phenyl)pent-4-enoate(18b)

Adapting a literature procedure (Jandeleit et al., U.S. Pat. No.8,168,617 (2012)), tert-butyl(3S)-3-(tert-butoxycarbonylamino)-5-(2-methyl-5-nitro-phenyl)pent-4-enoate(18b) is prepared from[(2S)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]-triphenyl-phosphoniumiodide (18a) (1.30 g, 2.0 mmol), 2-methyl-5-nitro-benzaldehyde (1b) (495mg, 3.0 mmol) (commercial or prepared in two steps from commercial2-methyl-5-nitro benzoic acid (i) BH₃.SMe₂, THF, reflux, ii) MnO₂, DCM,room temperature) as described in Example 1) with a commercial ˜1.0 Msolution of potassium tert-butoxide (KOtBu) (3.0 mL, 3.0 mmol) inanhydrous tetrahydrofuran (THF) (10 mL) at with gradual warming from 0°(ice bath) to room temperature for 24 hours. Aqueous work andpurification by silica gel column chromatography furnish the titlecompound (18b) as a mixture of geometric isomers ((E)/(Z)-isomers).

Step C: tert-Butyl(3R)-5-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-pentanoate(18c)

Following the General Procedure of Description 6 (Variant B), tert-butyl(3R)-5-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-pentanoate(18c) is prepared by catalytic reduction of tert-butyl(3S)-3-(tert-butoxycarbonylamino)-5-(2-methyl-5-nitro-phenyl)pent-4-enoate(18b) (813 mg, 2.0 mmol) in the presence of 10 wt-% palladium oncharcoal (Pd/C) containing ˜50 wt-% water (˜40 mg) in methanol (MeOH)(20 mL) and under an atmosphere of hydrogen (˜15 psi, H₂-balloon). Thecrude material, after filtration over Celite® 545, may be used directlyand without further isolation in the next step or may be purified bysilica gel chromatography to afford the title compound (18c).

Step D: tert-Butyl(3R)-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)pentanoate(18d)

Following the General Procedure of Description 7 (Variant C), tert-butyl(3R)-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)pentanoate(18d) is prepared from tert-butyl(3R)-5-(5-amino-2-methyl-phenyl)-3-(tert-butoxycarbonylamino)-pentanoate(18c) (757 mg, 2.0 mmol), 2-chloroacetaldehyde (˜50 wt-% in water, ˜7.87M) (1.27 mL, 10.0 mmol), and sodium cyanoborohydride (NaBH₃CN) (529 mgof 95% purity=503 mg, 8.0 mmol) in a mixture of methanol (MeOH) (10 mL)and 85 wt-% phosphoric acid (H₃PO₄) (3 mL). Aqueous work-up andpurification by silica gel column chromatography furnish the titlecompound (18d).

Step D:(3R)-3-Amino-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]pentanoicAcid (18)

Following the General Procedure of Description 9 (Variant B),(3R)-3-amino-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]pentanoicacid (18) is prepared from tert-butyl(3R)-5-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)pentanoate(18d) (504 mg, 1.0 mmol) in 2.0 N HCl in diethyl ether (2.0 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (18) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent of 1.0 Mhydrochloric acid (HCl).

Example 19(3R)-3-Amino-4-[5-(2-chloroethyl(chloromethyl)carbamoyl)oxy-2-methyl-phenyl]butanoicAcid (19)

Step A: tert-Butyl(3R)-3-(tert-butoxycarbonylamino)-4-(5-hydroxy-2-methyl-phenyl)-butanoate(19a)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (392 mg, 6.0 mmol) is activated with elemental iodine (I₂) (38 mg,0.15 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (19 μL,16 mg, 0.15 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (3 mL). The zinc insertion product is prepared from tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) (385 mg, 1.0mmol) in the presence of additional I₂ (38 mg, 0.15 mmol, 15 mol-%) andTMSCl (19 μL, 16 mg, 0.15 mmol, 15 mol-%). Following the GeneralProcedure of Description 15 (Part B), the zinc insertion product of (6c)is used in situ to cross couple with commercial 3-iodo-4-methyl-phenol(234 mg, 1.0 mmol) in the presence of tris(benzylideneacetone)dipalladium (Pd₂(dba)₃) (23 mg, 0.025 mmol, 2.5 mol-%) andtris(o-tolyl)phosphine (P(o-tol)₃) (30 mg, 0.10 mmol, 10 mol-%) inanhydrous degassed DMF (3 mL). Filtration, aqueous work-up, andpurification by silica gel column chromatography furnish the titlecompound (19a).

Step B: tert-Butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(chloromethyl)-carbamoyl)oxy-2-methyl-phenyl]butanoate(19b)

Adapting a literature known protocol (Fex, et al., U.S. Pat. No.3,299,104), tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(chloromethyl)-carbamoyl)oxy-2-methyl-phenyl]butanoate(19b) is prepared through carbamoylation of tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-(5-hydroxy-2-methyl-phenyl)-butanoate(19a) (731 mg, 2.0 mmol) with commercial N,N-bis(2-chloroethyl)carbamoylchloride (439 μL, 614 mg, 3.0 mmol) in anhydrous pyridine (15 mL) atabout 0° C. The reaction mixture is stirred with gradual warming to roomtemperature. The reaction is monitored by TLC and/or LC/MS tocompletion. Excess of the carbamoyl chloride is destroyed with crushedice. Aqueous work-up followed by purification through silica gel columnchromatography afford the title compound (19b).

Step C:(3R)-3-Amino-4-[5-(2-chloroethyl(chloromethyl)carbamoyl)oxy-2-methyl-phenyl]butanoicAcid (19)

Following the General Procedure of Description 9 (Variant B),(3R)-3-amino-4-[5-(2-chloroethyl(chloromethyl)carbamoyl)oxy-2-methyl-phenyl]butanoicacid (19) is prepared from tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethyl(chloromethyl)-carbamoyl)oxy-2-methyl-phenyl]butanoate(19b) (519 mg, 1.0 mmol) in 2.0 N HCl in diethyl ether (2.0 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (19) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent of 1.0 Mhydrochloric acid (HCl).

Example 20(3R)-3-Amino-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]butanoicAcid (20)

Step A: tert-Butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(hydroxymethyl)-2-nitro-phenyl]butanoate(20a)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (392 mg, 6.0 mmol) is activated with elemental iodine (I₂) (38 mg,0.15 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (19 μL,16 mg, 0.15 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (3 mL). The zinc insertion product is prepared from tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) (385 mg, 1.0mmol) in the presence of additional I₂ (38 mg, 0.15 mmol, 15 mol-%) andTMSCl (19 μL, 16 mg, 0.15 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part A), the zincinsertion product of (6c) is used in situ to cross couple withcommercial (3-bromo-4-nitro-phenyl)methanol (232 mg, 1.0 mmol) in thepresence of tris(benzylideneacetone) dipalladium (Pd₂(dba)₃) (23 mg,0.025 mmol, 2.5 mol-%) and tris(o-tolyl)phosphine (P(o-tol)₃) (30 mg,0.10 mmol, 10 mol-%) in anhydrous degassed DMF (3 mL). Filtration,aqueous work-up, and purification by silica gel column chromatographyfurnish the title compound (20a).

Step B: tert-Butyl(3R)-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]-3-(tert-butoxycarbonylamino)butanoate(20b)

Adapting a literature known protocol (Fex, et al., U.S. Pat. No.3,299,104), tert-butyl(3R)-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]-3-(tert-butoxycarbonylamino)butanoate(20b) is prepared through carbamoylation of tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(hydroxymethyl)-2-nitro-phenyl]butanoate(20a) (731 mg, 2.0 mmol) with commercial N,N-bis(2-chloroethyl)carbamoylchloride (439 μL, 614 mg, 3.0 mmol) in anhydrous pyridine (15 mL) atabout 0° C. The reaction mixture is stirred with gradual warming to roomtemperature. The reaction is monitored by TLC and/or LC/MS tocompletion. Excess of the carbamoyl chloride is destroyed with crushedice. Aqueous work-up followed by purification through silica gel columnchromatography afford the title compound (20b).

Step C:(3R)-3-Amino-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]butanoicAcid (20)

Following the General Procedure of Description 9 (Variant B),(3R)-3-amino-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]butanoicacid (20) is prepared from tert-Butyl(3R)-4-[5-[bis(2-chloroethyl)carbamoyloxymethyl]-2-nitro-phenyl]-3-(tert-butoxycarbonylamino)butanoate(20b) (578 mg, 1.0 mmol) in 2.0 N HCl in diethyl ether (2.0 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (20) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent of 1.0 Mhydrochloric acid (HCl).

Example 21(3R)-3-amino-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoicAcid (21)

Step A: tert-Butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoate(21a)

Adapting literature known protocols (Tercel, et al., J. Med. Chem. 1995,38, 1247-1252; Kirkpatrick, U.S. Pat. No. 5,602,278; Kirkpatrick, etal., Anti-Cancer Drugs, 1994, 5, 467-472; and Kirkpatrick, et al., U.S.Pat. No. 7,399,785), tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoate(21a) is prepared by adding 3-chloroperoxybenzoic acid (1.42 g, 80 wt-%,6.6 mmol) to a solution of tert-butyl(3R)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(6e) (2.43 g, 5.0 mmol) in dichloromethane (DCM) (30 mL) at about roomtemperature for about 2 h. The reaction is followed by TLC and/or LC/MSto completion. After quenching with a saturated aqueous solution ofsodium hydrogencarbonate (NaHCO₃), the reaction mixture is extractedwith DCM (3×). Further aqueous work-up and purification by silica gelcolumn chromatography yield the title compound (21a).

Step B:(3R)-3-amino-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoicacid (21)

Following the General Procedure of Description 9 (Variant B),(3R)-3-amino-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoicacid (21) is prepared from tert-butyl(3R)-3-(tert-butoxycarbonylamino)-4-[5-(2-chloroethoxy(2-chloroethyl)amino)-2-methyl-phenyl]butanoate(21a) (506 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2.0 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (21) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 224-[1-(Aminomethyl)-3-hydroxy-1-methyl-3-oxo-propyl]-N,N-bis(2-chloroethyl)-3-methyl-benzeneamineOxide (22)

Step A:3-[(2R)-4-tert-Butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]-N,N-bis(2-chloroethyl)-4-methyl-benzeneamineOxide (22a)

Adapting literature known protocols (Tercel, et al., J. Med. Chem. 1995,38, 1247-1252; and Kirkpatrick, et al., U.S. Pat. No. 7,399,785),peracetic acid (H₃CCO₃H) is freshly prepared by adding hydrogen peroxide(H₂O₂) (1.5 mL of a 35 wt-% aqueous solution, 14.0 mmol) dropwise toacetic anhydride (Ac₂O) (1.52 mL, 1.65 g, 16.0 mmol). When the reactionmixture is homogeneous, a solution of tert-butyl(3R)-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]-3-(tert-butoxycarbonylamino)butanoate(6e) (1.61 g, 3.29 mmol) in dichloromethane (DCM) (20 mL) is added withvigorous stirring at about room temperature for about 2 h. The reactionis followed by TLC and/or LC/MS to completion. The reaction is quenchedwith 2.0 N hydrochloric acid (HCl), and the aqueous layer separated andrepeatedly washed with DCM to the organic extracts are colorless. Theaqueous phase is evaporated to dryness under reduced pressure, driedover anhydrous sodium sulfate (Na₂SO₄), and partially reduced in volume.Diethyl ether (Et₂O) is added to separate the title compound3-[(2R)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]-N,N-bis(2-chloroethyl)-4-methyl-benzeneamineoxide (22a). The material may be purified by silica gel columnchromatography.

Step B:3-[(2R)-2-Amino-4-hydroxy-4-oxo-butyl]-N,N-bis(2-chloroethyl)-4-methyl-benzeneamineOxide (22)

Following the General Procedure of Description 9 (Variant B),3-[(2R)-2-amino-4-hydroxy-4-oxo-butyl]-N,N-bis(2-chloroethyl)-4-methyl-benzeneamineoxide (22) is prepared from3-[(2R)-4-tert-Butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]-N,N-bis(2-chloroethyl)-4-methyl-benzeneamineoxide (22a) (506 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl inEt₂O) (10 mL, 20 mmol) to yield the target compound (22) as an soliddihydrochloride salt (22.2HCl) after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 23(3R)-3-Amino-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]butanoic Acid (23)

Step A: Methyl2-[(2R)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]benzoate(23a)

Following the General Procedure of Description 15 (Part A), zinc dust(Zn) (392 mg, 6.0 mmol) is activated with elemental iodine (I₂) (38 mg,0.15 mmol, 15 mol-%) and trimethyl silylchloride (MeSiCl, TMSCl) (19 μL,16 mg, 0.15 mmol, 15 mol-%) in degassed anhydrous N,N-dimethylformamide(DMF) (3 mL). The zinc insertion product is prepared from tert-butyl(3S)-3-(tert-butoxycarbonylamino)-4-iodo-butanoate (6c) (385 mg, 1.0mmol) in the presence of additional I₂ (38 mg, 0.15 mmol, 15 mol-%) andTMSCl (19 μL, 16 mg, 0.15 mmol, 15 mol-%).

Following the General Procedure of Description 15 (Part B), the zincinsertion product is used in situ to cross couple with commercial methyl2-iodobenzoate (262 mg, 1.0 mmol) in the presence oftris(benzylideneacetone) dipalladium (Pd₂(dba)₃) (23 mg, 0.025 mmol, 2.5mol-%) and tris(o-tolyl)phosphine (P(o-tol)₃) (30 mg, 0.10 mmol, 10mol-%) in anhydrous degassed DMF (3 mL). Filtration, aqueous work-up,and purification by silica gel column chromatography furnish the titlecompound (23a).

Step B:2-[(2R)-4-tert-Butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]benzoicAcid (23b)

Adapting a literature known protocol (Dayal, et al., Steroids, 1990,55(5), 233-237), a reaction mixture of methyl2-[(2R)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]benzoate(23a) (1.97 g, 5.0 mmol) and commercial lithium hydroxide monohydrate(LiOH.H₂O) (420 mg, 10.0 mmol) in a mixture of water (10 mL) andmethanol (MeOH) (3 mL) is stirred at room temperature. The reaction ismonitored by TLC and/or LC/MS to completion. The solvent is partiallyremoved under reduced pressure using a rotary evaporator. Acidic aqueouswork-up and purification by silica gel column chromatography furnish thetitle compound2-[(2R)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]benzoicacid (23b) which may be used directly in the next step without furtherisolation and purification.

Step C: tert-Butyl(3R)-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]-3-(tert-butoxycarbonylamino)butanoate(23c)

Adapting a literature known protocol (Levi and Weed, U.S. Pat. No.3,235,594), to a reaction mixture of2-[(2R)-4-tert-butoxy-2-(tert-butoxycarbonylamino)-4-oxo-butyl]benzoicacid (23b) (759 mg, 2.0 mmol), N-hydroxysuccinimide (NHS, HOSu) (235 mg,2.2 mmol) in anhydrous acetonitrile (MeCN) (10 mL) is added soliddicyclohexylcarbodiimide (DCC) (433 mg, 2.1 mmol) in small portions atabout room temperature. The reaction mixture is stirred for about 12hours and the precipitated dicyclohexylurea side product is filtered offusing a Büchner funnel. The filtrate is treated with commercialdi-(2-chloroethyl)amine hydrochloride(2-chloro-N-(2-chloroethyl)ethanamine hydrochloride;HN(CH₂—CH₂—Cl)₂.HCl) (393 mg, 2.2 mmol) followed by neat triethylamine(Et₃N, TEA) (321 μL, 233 mg, 2.3 mmol). The reaction mixture is stirredfor about 12 hours at room temperature. The reaction is followed by TLCand/or LC/MS to completion. Acidic aqueous work-up and purification bysilica gel column chromatography furnish the title compound tert-butyl(3R)-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]-3-(tert-butoxycarbonylamino)butanoate(23c).

Step E: (3R)-3-Amino-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]butanoicAcid (23)

Following the General Procedure of Description 9 (Variant B),(3R)-3-amino-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]butanoic acid (23)is prepared from tert-butyl(3R)-4-[2-[bis(2-chloroethyl)carbamoyl]phenyl]-3-(tert-butoxycarbonylamino)butanoate(23c) (504 mg, 1.0 mmol) in 2 N HCl in diethyl ether (2 N HCl in Et₂O)(10 mL, 20 mmol) to yield the target compound (23) as an soliddihydrochloride salt after evaporation of the solvents andlyophilization from an aqueous solution. The material may be furtherpurified by preparative HPLC followed by lyophilization. Optionally, thelyophilization is conducted in the presence of 1 equivalent or an excessof 1.0 M hydrochloric acid (HCl).

Example 24 LAT1 Uptake Inhibition Assays

The ability of compounds to interact with LAT1 was measured using aradiolabeled competition uptake assay with [³H]-Gabapentin (GP) in96-well plates with LLCPK cells conditionally expressing hLAT1. 5×10⁴cells/well were plated in white, clear bottom plates in the presence orabsence of tetracycline or doxcycline to induce hLAT1 expression. Thenext day, cells were treated with butyrate to stimulate additional hLAT1expression. On the third day, the cells were washed and then incubatedwith 50,000 cpm of [³H]-GP in PBS in the presence or absence of 1 mM oftest compound in at least triplicate for 15 min. At end of the assaytime, the incubation solution was removed, and the plates were washedthree times with 100 μl of ice-cold PBS. 150 μl of scintillation fluidwas added to each well, and the radioactivity retained within the cellswas measured on a 96-well scintillation counter. The data are expressedas a percent of specific [³H]-GP uptake. Unlabeled GP and other largeamino acids (phenylalanine and leucine) were used as controls.

The ability of various compounds to interact with LAT1 was assessed bymeasuring the inhibition of [³H]-GP uptake into LAT1-expressing cells inthe presence of 1 mM test compound. Unlabeled GP and phenylalanine (Phe)and leucine (Leu) were used as controls. After incubation for 15 min,cells were washed, scintillation fluid added, and cell-boundradioactivity determined in a scintillation counter. Data are expressedas a percent of specific GP uptake.

The specific uptake of radiolabeled gabapentin into LAT1-expressingcells was inhibited by 1 mM of unlabeled gabapentin, phenylalanine,leucine, and the compounds of Examples 1-4. Treatment with gabapentin,phenylalanine, leucine, and the compound of Example 3 resulted inspecific uptake of less than 10%. The compounds of Examples 1, 2, and 4resulted in specific uptake of greater than 20% but less than 50% atthis concentration. The specific uptake of radiolabeled gabapentin inthe absence of any compound was 100%.

Example 25 LAT1-Specific In Vitro Cytotoxicity Assays

The LAT1-specific in vitro cytotoxicity of compounds was assessed byusing a modified clonigenic assay in 96-well plates with LLCPK cellsconditionally expressing hLAT1. 1000 cells/well were plated in clearbottom plates in the presence or absence of tetracycline or doxcyclineto induce hLAT1 expression. The next day, cells were treated withbutyrate to stimulate additional hLAT1 expression. On the third day,cells were washed and incubated with various concentrations of testcompounds in PBS buffer in at least quadruplicate for 30 minutes. At theend of the treatment, test compounds were removed and growth media wasadded to the cells. Clonal populations were allowed to grow until thecontrol wells (mock treatment) were near confluency (7 to 10 days). Cellgrowth was quantified by fixing and staining the cells post-wash withcrystal violent in glutaraldehye, washing away unadhered dye,solubilizing the stained cells in acetic acid and monitoring absorbanceat 530 nm. Data from each test concentration were expressed as thepercent of live, mock-treated controls (% surviving cells). LAT1specificity was determined by the differential toxicity in cells induced(LAT1+) vs. non-induced (no LAT1) to express hLAT1. Melphalan, aN-mustard compound, was used as a control.

The LAT1-specific cytotoxicity of various compounds was assessed bytreating cells expressing or not expressing LAT1 with 3 μM of testcompound for 30 min. Melphalan was used as a control compound. Followingtreatment, cells were washed and growth media was added. Surviving cellswere allowed to proliferate for 7-10 days, and then stained andquantified. Results were expressed as the percent of untreated cells (%surviving cells).

The percent surviving cells for melphalan and the compound of Example 2was about the same in cells expressing LAT1 and in cells not expressingLAT1. The percent surviving cells for the compounds of Examples 1, 3,and 4 was significantly reduced by at least 25% in cells expressing LAT1compared to cells not expressing LAT1.

The in vitro cytotoxicity of the two enantiomers of QBS10072 wasassessed by treating LAT1-expressing cells with various concentrationsof the S (solid circles) or the R (open circles) isomer for 30 min.Following treatment, cells were washed and growth media was added.Surviving cells were allowed to proliferate for 7-10 days, and thenstained and quantified. Results were expressed as the percent ofuntreated cells (% surviving cells) and graphed vs. test concentration.

The in vitro cytotoxicity of the two single enantiomers of Example 3 wasassessed by treating LAT1-expressing cells with various concentrationsof the S (solid circles) or the R (open circles) isomer for 30 min.Following treatment, cells were washed and growth media was added.Surviving cells were allowed to proliferate for 7-10 days, and thenstained and quantified. Results were expressed as the percent ofuntreated cells (% surviving cells) and graphed vs. test concentration.

Example 26 In Vivo Tumor Growth Suppression Assays

The ability to suppress the growth of tumors in vivo was measured usinga B16 efficacy model (Kato, et al., Cancer Res., 1994, 54, 5143-5147).Briefly, the hind flank of C57BL/6 mice were injected with 5×10⁵ B16melanoma cells subcutaneously. Once the tumors reached 40 mm³, animalswere separated into various treatment arms (n=5) and dosed IP daily withvehicle or test compound (5 and 10 mg/kg) for 12 days. Tumor sizes weremonitored every third day for up to three weeks. Melphalan was used as acontrol compound (2.5 mg/kg). The results are presented in Table 1.

TABLE 1 Tumor Suppression by QBS Compounds in vivo Tumor Growth (%Control) Treatment End of dosing 5 days post-dosing Vehicle 100 100QBS10072 11 11 Melphalan 33 56

Finally it should be noted that there are alternative ways ofimplementing the embodiments disclosed herein. Accordingly, the presentembodiments are to be considered as illustrative and not restrictive,and the claims are not to be limited to the details given herein, butmay be modified within the scope and equivalents thereof.

What is claimed is:
 1. A method of treating cancer in a patientcomprising administering to a patient in need of such treatment atherapeutically effective amount of(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (5), or a pharmaceutically acceptable salt thereof, wherein thecancer comprises glioblastoma, triple negative breast cancer, melanoma,prostate cancer, or multiple myeloma.
 2. The method of claim 1, whereinadministering comprises administering a pharmaceutical composition,wherein the pharmaceutical composition comprises: a therapeuticallyeffective amount of(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (5) or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable vehicle.
 3. The method of claim 1, whereinthe cancer comprises cancerous tissue that over-expresses the LAT1/4F2hctransporter compared to surrounding non-cancerous tissue.
 4. The methodof claim 1, wherein the cancer is known to be treated by administeringan alkylating agent.
 5. The method of claim 1, wherein the brain cancercomprises glioblastoma.
 6. The method of claim 1, wherein the breastcancer comprises triple negative breast cancer.
 7. The method of claim1, wherein the skin cancer comprises melanoma.
 8. The method of claim 1,wherein the cancer comprises prostate cancer.
 9. The method of claim 1,wherein the cancer comprises multiple myeloma.
 10. The method of claim1, wherein administering comprises administering(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (5) in combination with at least one additional therapeutic agent.11. The method of claim 1, wherein administering comprises administering(3S)-3-amino-4-[5-[bis(2-chloroethyl)amino]-2-methyl-phenyl]butanoicacid (5) in combination with at least one additional chemotherapeuticagent.